The present invention provides a highly sensitive method of diagnosing inflammatory bowel disease (ibd) in an individual. The method includes the steps of isolating a sample from the individual; determining by non-histological means whether the sample is positive for anti-neutrophil cytoplasmic antibodies (anca); determining whether the sample is positive for anti-Saccharomyces cerevisiae immunoglobulin A (asca-IgA); determining whether the sample is positive for anti-Saccharomyces cerevisiae immunoglobulin G (asca-IgG); and diagnosing the individual as having ibd when the sample is positive for anca, asca-IgA or asca-IgG, and diagnosing the individual as not having ibd when the sample is negative for anca, asca-IgA and asca-IgG, provided that the method does not include histological analysis of neutrophils.
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23. A highly efficient method of analyzing multiple samples for ibd, comprising the steps of:
(a) first assaying all samples by non-histological means for the presence or absence of anca; (b) next assaying only anca-negative samples for the presence or absence of asca-IgA; (c) next assaying only anca-negative and asca-IgA-negative samples for the presence or absence of asca-IgG, wherein the presence of anca, asca-IgA or asca-IgG in a sample is indicative of ibd and wherein the absence of pANCA, asca-IgA and asca-IgG is indicative of the absence of ibd.
1. A highly sensitive method of diagnosing inflammatory bowel disease (ibd) in an individual, comprising the steps of:
(a) isolating a sample from said individual; (b) determining by non-histological means whether said sample is positive for anti-neutrophil cytoplasmic antibodies (anca); (c) determining whether said sample is positive for anti-Saccharomyces cerevisiae immunoglobulin A (asca-IgA); (d) determining whether said sample is positive for anti-Saccharomyces cerevisiae immunoglobulin G (asca-IgG); and (e) diagnosing said individual as having ibd when said sample is positive for anca, asca-IgA or asca-IgG, and diagnosing said individual as not having ibd when said sample is negative for anca, asca-IgA and asca-IgG, provided that said method does not include histological analysis of neutrophils.
4. A highly sensitive method of diagnosing ibd in an individual, comprising the steps of:
(a) isolating a sample from said individual; (b) determining by non-histological means whether said sample has an anca level above an anca cut-off value (x); (c) determining whether said sample has an asca-IgA level above an asca-IgA cut-off value (y); (d) determining whether said sample has an asca-IgG level above an asca-IgG cut-off value (Z); and (e) diagnosing said individual as having ibd when said anca level is above x, said asca-IgA level is above y, or said asca-IgG level is above Z, and diagnosing said individual as not having ibd when said anca level is below x, said asca-IgA level is below y. and said asca-IgG value is below Z, wherein x, y, and Z are independently selected to achieve an optimized clinical parameter selected from the group consisting of: sensitivity, specificity, negative predictive value, positive predictive value and overall agreement, provided that said method does not include histological analysis of neutrophils.
17. A highly sensitive method of diagnosing ibd in an individual, comprising the steps of:
(a) isolating a sample from said individual; (b) contacting an appropriate dilution of said sample with antigen specific for anca under conditions suitable to form a first complex of anca and antigen specific for anca to determine by non-histological means the amount of said first complex; (c) contacting an appropriate dilution of said sample with antigen specific for asca under conditions suitable to form a second complex of asca and antigen specific for asca; (d) contacting said second complex with anti-immunoglobulin A antibody to determine the amount of asca-IgA containing second complex; (e) contacting said second complex with anti-immunoglobulin G antibody to determine the amount of asca-IgG containing second complex; (f) diagnosing said individual as having ibd when the amount of first complex formed is greater than an anca cut-off value (x), the amount of IgA-containing second complex formed is greater than an asca-IgA cut-off value (y), or the amount of IgG-containing second complex formed is greater than an asca-IGG cut-off value (Z), and diagnosing said individual as not having ibd when the amount of first complex formed is less than x, the amount of IgA-containing second complex formed is less than y, and the amount of IgG-containing second complex formed is less than Z, wherein x, y, and Z are independently selected to achieve an optimized clinical parameter selected from the group consisting of: sensitivity, specificity, negative predictive value, positive predictive value and overall agreement, provided that said method does not include histological analysis of neutrophils.
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The invention relates generally to the fields of inflammatory bowel disease and immunology and more specifically to serological methods for distinguishing inflammatory bowel disease from other disorders.
Inflammatory bowel disease (IBD), which occurs world-wide and afflicts millions of people, is the collective term used to describe two gastrointestinal disorders of unknown etiology: Crohn's disease (CD) and ulcerative colitis (UC). IBD together with irritable bowel syndrome (IBS) will affect one-half of all Americans during their lifetime, at a cost of greater than $2.6 billion dollars for IBD and greater than $8 billion dollars for IBS. A primary determinant of these high medical costs is the difficulty of diagnosing digestive diseases. The cost of IBD and IBS is compounded by lost productivity, with persons suffering from these disorders missing at least 8 more days of work annually than the national average.
Inflammatory bowel disease has many symptoms in common with irritable bowel syndrome, including abdominal pain, chronic diarrhea, weight loss and cramping, making definitive diagnosis extremely difficult. Of the 5 million people suspected of suffering from IBD in the U.S., only 1 million are diagnosed as such. The difficulty in differentially diagnosing IBD and IBS hampers early and effective treatment of these diseases. Thus, there is a need for rapid and sensitive testing methods for definitively distinguishing IBD from IBS.
Progress has been made in precisely diagnosing, in many cases, Crohn's disease and ulcerative colitis. However, current methods for diagnosing an individual as having Crohn's disease or ulcerative colitis, while highly specific, are relatively costly, requiring labor intensive immunofluorescence assays and careful analysis of cell staining patterns. Although these costly assays are easily justified for those individuals previously diagnosed with or strongly suggested to have IBD, a less expensive but highly sensitive alternative would be advantageous for first determining if an individual has inflammatory bowel disease at all. Such a highly sensitive primary screening assay would provide physicians with an inexpensive means for rapidly distinguishing individuals with IBD from those having IBS, thereby facilitating earlier and more appropriate therapeutic intervention and minimizing uncertainty for patients and their families. If desired, such a primary screening assay could be combined with a subsequent, highly specific assay for determining if an individual diagnosed with IBD has Crohn's disease or ulcerative colitis.
Unfortunately, such a highly sensitive and inexpensive primary screening assay for distinguishing IBD from other digestive diseases presenting with similar symptoms is currently not available. Thus, there is a need for a method of rapidly diagnosing inflammatory bowel disease at a very early stage of disease progression. The present invention satisfies this need and provides related advantages as well.
The present invention provides a highly sensitive method of diagnosing inflammatory bowel disease (IBD) in an individual. The method includes the steps of isolating a sample from the individual; determining by non-histological means whether the sample is positive for anti-neutrophil cytoplasmic antibodies (ANCA); determining whether the sample is positive for anti-Saccharomyces cerevisiae immunoglobulin A (ASCA-IgA); determining whether the sample is positive for anti-Saccharomyces cerevisiae immunoglobulin G (ASCA-IgG); and diagnosing the individual as having IBD when the sample is positive for ANCA, ASCA-IgA or ASCA-IgG, and diagnosing the individual as not having IBD when the sample is negative for ANCA, ASCA-IgA and ASCA-IgG, provided that the method does not include histological analysis of neutrophils. In a method of the invention, ANCA, ASCA-IgA or ASCA-IgG positivity can be conveniently determined, for example, using an immunoassay.
The present invention further provides a highly sensitive method of diagnosing IBD in an individual. This method of the invention includes the steps of isolating a sample from the individual; determining by non-histological means whether the sample has an ANCA level above an ANCA cut-off value (X); determining whether the sample has an ASCA-IgA level above an ASCA-IgA cut-off value (Y); determining whether the sample has an ASCA-IgG level above an ASCA-IgG cut-off value (Z); and diagnosing the individual as having IBD when the ANCA level is above X, the ASCA-IgA level is above Y, or the ASCA-IgG level is above Z, and diagnosing the individual as not having IBD when the ANCA level is below X, the ASCA-IgA level is below Y, and the ASCA-IgG value is below Z, where X, Y, and Z are independently selected to achieve optimized sensitivity, specificity, negative predictive value, positive predictive value or overall agreement, provided that the method does not include histological analysis of neutrophils.
In a highly sensitive method of diagnosing IBD provided by the present invention, X, Y and Z can be independently selected such that, for example, the sensitivity of diagnosing an individual with IBD is at least about 70%, and can be selected such that, additionally, the specificity of diagnosing an individual with IBD is 30-60%. In addition, X, Y and Z can be independently selected such that the sensitivity of diagnosing an individual with IBD is at least about 70%, the specificity of diagnosing an individual with IBD is 30-60%, and the negative predictive value in a population having an IBD disease prevalence of about 15% is at least about 90% and can be, for example, at least about 95%.
Furthermore, X, Y and Z can be independently selected such that the sensitivity of diagnosing an individual with IBD is at least about 90%, and can be selected such that, additionally, the specificity of diagnosing an individual with IBD is 20-60%. If desired, X, Y and Z can be independently selected such that the sensitivity of diagnosing an individual with IBD is at least about 90%, the specificity of diagnosing an individual with IBD is 20-60%, and the negative redictive value in a population having an IBD disease revalence of about 15% is at least about 90%. The negative predictive value can be, for example, at least about 95%. In addition, X, Y and Z can be independently selected such that, for example, the sensitivity of diagnosing an individual with IBD is about 90%, the specificity is about 37%, and the negative predictive value in a population having an IBD disease prevalence of about 15% is at least about 95%. In one embodiment, X can be selected to be 0.7 multiplied by two standard deviations above the background value of ANCA-negative UC sera, Y can be selected to be 12 ELISA units, and Z can be selected to be 60 ELISA units.
In a method of the invention for diagnosing inflammatory bowel disease, the ANCA, ASCA-IgA and ASCA-IgG levels can be determined using, for example, a serum sample or saliva sample. ANCA levels can be determined using an antigen specific for ANCA such as fixed neutrophils, and ASCA-IgA and ASCA-IgG levels can be determined using an antigen specific for ASCA such as yeast cell wall phosphopeptidomannan (PPM), which can be prepared, for example, from strain ATCC #38926.
The invention additionally provides a highly efficient method of analyzing multiple samples for IBD by first assaying all samples for the presence or absence of ANCA; next assaying only ANCA-negative samples for the presence or absence of ASCA-IgA; and next assaying only ANCA-negative and ASCA-IgA-negative samples for the presence or absence of ASCA-IgG, where the presence of pANCA, ASCA-IgA or ASCA-IgG in a sample is indicative of IBD and where the absence of ANCA, ASCA-IgA and ASCA-IgG is indicative of the absence of IBD. In such a method of the invention, the presence of ANCA, ASCA-IgA and ASCA-IgG can be conveniently determined, for example, using an immunoassay.
FIG. 1 shows the IBD First Step central composite design.
The present invention is directed to the discovery that three enzyme-linked immunosorbent assays (ELISAs) can be combined without immunofluorescence or other histological analysis of neutrophils to diagnose inflammatory bowel disease (IBD) with high sensitivity. In particular, as disclosed in Example II, an ELISA assay for anti-neutrophil cytoplasmic antibodies (ANCA), an ELISA assay for anti-Saccharomyces cerevisiae immunoglobulin A (ASCA-IgA) and an ELISA assay for anti-Saccharomyces cerevisiae immunoglobulin G (ASCA-IgG) were combined to produce a highly sensitive means of distinguishing individuals having either Crohn's disease (CD) or ulcerative colitis (UC) from other individuals, such as those having irritable bowel syndrome (IBS). Such a method does not include labor intensive immunofluorescence analysis of fixed neutrophils or other neutrophil histological analysis. Thus, the methods of the invention provide a rapid and sensitive means of differentiating individuals having either CD or UC from those who do not have IBD. The methods of the invention can be used alone to rule out inflammatory bowel disease in an individual suspected of having the disease, or, when positive for diagnosis of IBD, can be used in combination with a subsequent assay that specifically differentiates CD from UC.
Thus, the present invention provides a highly sensitive method of diagnosing IBD in an individual. The method includes the steps of isolating a sample from the individual; determining by non-histological means whether the sample is positive for ANCA; determining whether the sample is positive for ASCA-IgA; determining whether the sample is positive for ASCA-IgG; and diagnosing the individual as having IBD when the sample is positive for ANCA, ASCA-IgA or ASCA-IgG, and diagnosing the individual as not having IBD when the sample is negative for ANCA, ASCA-IgA and ASCA-IgG, provided that the method does not include histological analysis of neutrophils. In a method of the invention, ANCA, ASCA-IgA and ASCA-IgG positivity can be determined, for example, using an immunoassay.
As used herein, the term "inflammatory bowel disease" is synonymous with "IBD" and is a collective term referring to both Crohn's disease and ulcerative colitis. Thus, an individual having either Crohn's disease or ulcerative colitis is defined herein as having IBD. Conversely, an individual having neither ulcerative colitis nor Crohn's disease does not have IBD as defined herein. The term "inflammatory bowel disease" distinguishes Crohn's disease and ulcerative colitis from all other disorders, syndromes or abnormalities of the gastroenterological tract including irritable bowel syndrome.
The methods of the invention for diagnosing IBD involve determining whether a sample is positive for anti-neutrophil cytoplasmic antibodies (ANCA). Anti-neutrophil cytoplasmic antibodies that produce a perinuclear staining pattern (pANCA) are elevated in 60-80% of UC patients and less frequently in CD and other disorders of the colon. Serum titers of ANCA are elevated in UC patients regardless of clinical status and, thus, do not reflect disease activity. High levels of serum ANCA also persist in UC patients five years post-colectomy. Although pANCA is found only very rarely in healthy adults and children, healthy relatives of UC patients have an increased frequency of pANCA, indicating that pANCA may be an immunogenetic susceptibility marker. ANCA reactivity is also present in a small portion of patients with Crohn's disease. The reported prevalence in CD varies, with most studies reporting that 10 to 30% of CD patients express ANCA (Saxon et al., J. Allergy Clin. Immunol. 86:202-210 (1990); Cambridge et al., Gut 33:668-674 (1992); Pool et al., Gut 3446-50 (1993); and Brokroelofs et al., Dig. Dis. Sci. 39:545-549 (1994)).
As used herein, the term "anti-neutrophil cytoplasmic antibody" is synonymous with "ANCA" and means antibodies to cytoplasmic components of a neutrophil. ANCA, such as serum or saliva ANCA, can be detected using an enzyme-linked immunosorbent assay with alcohol-fixed neutrophils, for example (see Example I). As disclosed herein, ANCA activity is divided into several broad categories: perinuclear to nuclear staining or cytoplasmic staining with perinuclear highlighting (pANCA); cytoplasmic neutrophil staining without perinuclear highlighting (cANCA); and diffuse staining with speckling across the entire neutrophil (SAPPA). The term ANCA, as used herein, encompasses all varieties of anti-neutrophils cytoplasmic reactivity, including pANCA, cANCA and SAPPA. Similarly, the term "ANCA" encompasses all immunoglobulin isotypes including, for example, immunoglobulin A and G.
The determination of whether a sample is positive for ANCA using non-histological means is made using antigen specific for ANCA. Such an antigen specific for ANCA can be, for example, whole fixed neutrophils; an unpurified or partially purified neutrophil extract; a purified UC pANCA antigen such as a purified protein, protein fragment or synthetically produced peptide; an anti-ANCA idiotypic antibody; or the like. Particularly useful antigens specific for ANCA are peptides, which can be chemically synthesized or expressed on the surface of phage. Purified antigens specific for ANCA can be, for example, histone H1, or an ANCA-reactive fragment of histone H1, as described in U.S. Pat. No. 6,074,835 now U.S. Pat. No. 6,074,835; an ulcerative colitis pANCA secretory vesicle antigen or an ANCA-reactive fragment thereof, as described in U.S. application Ser. No. 08/804,106; or a microbial UC pANCA antigen, such as a histone H1-like antigen, porin antigen, Bacteroides antigen, or ANCA-reactive fragment thereof, as described in U.S. Pat. No. 6,033,864 now U.S. Pat. No. 6,033,864. One skilled in the art understands that additional antigens specific for ANCA, including antigenic fragments and ANCA-reactive peptides, can be identified, for example, using a representative UC pANCA monoclonal antibody, such as one described in U.S. application Ser. No. 08/472,688, now abandoned.
In the methods of the invention, a sample to be analyzed is obtained from the individual to be diagnosed. The term "sample," as used herein, means any biological specimen obtained from an individual that contains antibodies. A sample can be, for example, whole blood, plasma, saliva or other bodily fluid or tissue having antibodies, preferably a serum sample. Preferably, although not necessarily, a sample contains both ANCA and ASCA antibodies. The use of a serum sample is described in Example I; the use of other samples, such as saliva and urine samples, is well known in the art (see, for example, Hashida et al., J. Clin. Lab. Anal. 11:267-86 (1997), which is incorporated by reference herein). One skilled in the art understands that samples such as serum samples can be diluted prior to analysis of ANCA, ASCA-IgA and ASCA-IgG content.
The methods of the invention for diagnosing IBD also involve determining whether a sample is positive for immunoglobulin A anti-Saccharomyces cerevisiae antibodies (ASCA-IgA) or immunoglobulin G anti-Saccharomyces cerevisiae antibodies (ASCA-IgG). Previous reports indicate that such antibodies can be elevated in patients having Crohn's disease, although the nature of the S. cerevisiae antigen supporting the specific antibody response in CD is unknown (Sendid et al., Clin. Diag. Lab. Immunol. 3:219-226 (1996), which is incorporated herein by reference). ASCA may represent a response against yeasts present in common food or drink or a response against yeasts that colonize the gastrointestinal tract. Studies with periodate oxidation have shown that the epitopes recognized by ASCA in CD patient sera contain polysaccharides. Oligomannosidic epitopes are shared by a variety of organisms including different yeast strains and genera, filamentous fungi, viruses, bacteria and human glycoproteins. Thus, mannose-induced antibody responses in CD may represent a response against a pathogenic yeast organism or against a cross-reactive oligomannosidic epitope present, for example, on a human glycoprotein autoantigen. Regardless of the nature of the antigen, elevated levels of serum ASCA are believed to be a differential marker for Crohn's disease, with only low levels of ASCA reported in UC patients (Sendid et al., supra, 1996).
As used herein, the term "anti-Saccharomyces cerevisiae immunoglobulin A" is synonymous with "ASCA-IgA" and refers to antibodies of the immunoglobulin A isotype that react specifically with S. cerevisiae. Similarly, the term "anti-Saccharomyces cerevisiae immunoglobulin G" is synonymous with "ASCA-IgG" and refers to antibodies of the immunoglobulin G isotype that react specifically with S. cerevisiae. The determination of whether a sample is positive for ASCA-IgA or ASCA-IgG is made using an antigen specific for ASCA. Such an antigen can be any antigen or mixture of antigens that is bound specifically by immunoglobulin A ASCA or immunoglobulin G ASCA. Although ASCA antibodies were initially characterized by their ability to bind S. cerevisiae, those of skill in the art will understand that an antigen that is bound specifically by ASCA can be obtained from S. cerevisiae, or can be obtained from a variety of other sources so long as the antigen is capable of binding specifically to ASCA antibodies. Accordingly, exemplary sources of an antigen specific for ASCA, which can be used to determine whether a sample is positive for ASCA-IgA or ASCA-IgG, include whole killed yeast cells, such as Saccharomyces or Candida cells; yeast cell wall phosphopeptidomannan (PPM); oligomannosides; neoglycolipids; anti-ASCA idiotypic antibodies; and the like. As described above, different species and strains of yeast, including Saccharomyces, can be an antigen specific for ASCA useful for determining whether a sample is positive for ASCA-IgA or ASCA-IgG. For example, S. cerevisiae strain Su1, Su2, CBS 1315 or BM 156, or Candida albicans strain VW32, can be used as an antigen specific for ASCA in a method of the invention.
Preparations of yeast cell wall mannans, or phosphopeptidomannans (PPM), are can be used to determine if a sample is positive for ASCA-IgA or ASCA-IgG. Such water soluble surface antigens can be prepared by appropriate extraction techniques, including autoclaving as described in Example I, or can be obtained commercially (see Lindberg et al., Gut 33:909-913 (1992), which is incorporated herein by reference). The acid stable fraction of yeast cell wall PPM also can be useful in the methods of the invention (Sendid et al., supra, 1996). An exemplary PPM that is useful in determining whether a sample is positive for ASCA-IgA or ASCA-IgG is derived from S. cerevisiae strain ATCC #38926.
Purified oligosaccharide antigens, such as oligomannosides, also can be useful in determining whether a sample is positive for ASCA-IgA or ASCA-IgG in a method of the invention. For use herein, the purified oligomannoside antigens are preferably converted into neoglycolipids as described in Faille et al., Eur. J. Microbiol. Infect. Dis. 11:438-446 (1992). One skilled in the art understands that the reactivity of such an oligomannoside antigen with ASCA can be optimized by varying the mannosyl chain length (Frosh et al., Proc Natl. Cad. Sci. USA, 82:1194-1198 (1985)); the anomeric configuration (Fukazawa et al., In E. Kurstak (ed.), Immunology of Fungal Disease, Marcel Dekker Inc., New York, pp. 37-62 (1989); Nishikawa et al, Microbiol. Immunol., 34:825-840 (1990); Poulain et al., Eur. J. Clin. Microbiol, 23:46-52 (1993); Shibata et al., Arch. Biochem. Biophys., 243:338-348 (1985); and Trinel et al., Infect. Immun., 60:3845-3851 (1992)); or the position of the linkage (Kikuchi et al., Planta, 190:525-535 (1993)). Each of the foregoing references are incorporated herein by reference in their entirety.
An antigen specific for ASCA useful in determining whether a sample is positive for ASCA-IgA or ASCA-IgG can be, for example, an oligomannoside which includes the mannotetraose Man(1→3)Man(1→2) Man(1→2)Man. Such an oligomannoside can be purified from PPM as described in Faille et al., supra, 1992. An exemplary neoglycolipid which is an antigen specific for ASCA can be constructed by releasing the oligomannoside from its respective PPM and subsequently coupling the released oligomannoside to 4-hexadecylaniline or the like.
Prior to the present invention, ANCA and ASCA analysis have been combined in order, for example, to increase the specificity of an assay for differentiating UC from Crohn's disease or to determine clinical subtypes of CD (Quinton et al., Gastroenterol. 112: A1066 (1997); Seidman et al., Gastroenterol. 112: A1087 (1997); and Vasiliauskas et al., Gastroenterol. 112: A1112 (1997)). In contrast, the methods of the present invention, which are of high sensitivity, are directed to determining if an individual has either UC or CD but do not distinguish between the two diseases. Thus, the methods of the invention are useful, for example, to sensitively distinguish between IBD and other digestive disorders such as irritable bowel syndrome and infectious digestive diseases and, when positive for IBD, can be used in conjunction, if desired, with a subsequent specific assay in order to precisely determine whether the individual with IBD has UC or CD. Furthermore, previous studies in which ANCA analysis has been combined with analysis of ASCA-IgA and ASCA-IgG have additionally included immunofluorescence of fixed neutrophils in order to determine the type of ANCA reactivity present (see Quinton et al., supra, 1997; Seidman et al., supra, 1997; Dubinsky et al., supra, 1997; and Vasiliauskas et al., supra, 1997). In contrast to these studies, the methods of the present invention explicitly exclude histological analysis of neutrophils.
Thus, the present invention is directed to a highly sensitive method of diagnosing inflammatory bowel disease, which does not include histological analysis of neutrophils. As used herein, the term "histological analysis of neutrophils" means any technique revealing the structure of a neutrophilic cell using staining or microscopy. Histological analysis, which encompasses techniques such as immunocytochemistry and indirect immunofluorescence, as well as other methods involving microscopy, is explicitly excluded from the present invention. In contrast, an enzyme-linked immunosorbent assay (ELISA), in which neutrophil reactivity is analyzed by means of a detectable secondary antibody that generates a quantitative signal, does not involve microscopy or other analysis of cell structure and, therefore, is not "histological analysis of neutrophils" as defined herein.
As further disclosed herein, three ELISA cut-off values for determining if a sample is positive or negative for ANCA ("X"), ASCA-IgA ("Y") and ASCA-IgG ("Z") were simultaneously varied using Factorial Design Optimization to achieve a desired degree of sensitivity (Example II). Using this approach, cooperative interactions among the ANCA, ASCA-IgA and ASCA-IgG cut-off values were identified. For example, particular ANCA, ASCA-IgA and ASCA-IgG cut-off values were determined to diagnose an individual with IBD with greater than about 90% sensitivity, which is a greater than 90% probability that an individual having IBD by colonoscopic, radiologic and/or histologic criteria would be diagnosed as such. Thus, the present invention provides a method of diagnosing inflammatory bowel disease with a greater sensitivity than previously available. Similarly, using Factorial Design Optimization, for example, other ANCA, ASCA-IgA and ASCA-IgG cut-off values can be determined which provide a clinically useful sensitivity, specificity, negative predictive value, positive predictive value or overall agreement for a particular patient population. If desired, one can select the ANCA, ASCA-IgA and ASCA-IgG cut-off values "X," "Y," and "Z" to give a desired sensitivity combined with, for example, a desired specificity and negative predictive value.
The present invention therefore provides a highly sensitive method of diagnosing IBD in an individual by isolating a sample from the individual; determining by non-histological means whether the sample has an ANCA level above an ANCA cut-off value (X); determining whether the sample has an ASCA-IgA level above an ASCA-IgA cut-off value (Y); determining whether the sample has an ASCA-IgG level above an ASCA-IgG cut-off value (Z); and diagnosing the individual as having IBD when the ANCA level is above X, the ASCA-IgA level is above Y, or the ASCA-IgG level is above Z, and diagnosing the individual as not having IBD when the ANCA level is below X, the ASCA-IgA level is below Y, and the ASCA-IgG value is below Z, where X, Y, and Z are independently selected to achieve an optimized sensitivity, specificity, negative predictive value, positive predictive value or overall agreement, provided that the method does not include histological analysis of neutrophils.
As used herein, the term "X" refers to an ANCA cut-off value, against which an experimental ANCA sample value is compared. Similarly, as used herein, the term "Y" refers to an ASCA-IgA cut-off value, against which an experimental ASCA-IgA value is compared. The term "Z," as used herein, refers to an ASCA-IgG cut-off value, against which an experimental ASCA-IgG cut-off value is compared. As disclosed herein, when an ANCA level is above X, or an ASCA-IgA level is above Y, or an ASCA-IgG level is above Z, an individual is diagnosed as having IBD.
The clinical parameters of sensitivity, specificity, negative predictive value, positive predictive value and overall agreement are calculated using true positives, false positives, false negatives and true negatives. A "true positive" sample is a sample positive for IBD according to colonoscopic, radiologic and/or histologic analysis, which is also diagnosed positive according to a method of the invention. A "false positive" sample is a sample negative for IBD by colbnoscopic, radiologic and/or histologic analysis, which is diagnosed positive according to a method of the invention. Similarly, a "false negative" is a sample positive for IBD by colonoscopic, radiologic and/or histologic analysis, which is diagnosed negative according to a method of the invention. A "true negative" is a sample negative for IBD by colonoscopic, radiologic and/or histologic analysis, and also negative for IBD according to a method of the invention. See, for example, Motulsky (Ed.), Intuitive Biostatistics New York: Oxford University Press (1995), which is incorporated herein by reference.
As used herein, the term "sensitivity" means the probability that a laboratory method is positive in the presence of IBD. Sensitivity is calculated as the number of true positive results divided by the sum of the true positives and false negatives. Sensitivity essentially is a measure of how well a method correctly identifies those with disease. In a method of the invention, the ANCA, ASCA-IgA, and ASCA-IgG values can be selected such that the sensitivity of diagnosing an individual is at least about 60%, and can be, for example, at least about 65%, 70%, 75%, 80%, 85%, 90% or 95%. As illustrated in Example II, the maximum sensitivity of diagnosing IBD using a method of the invention is about 96.5%. A method of diagnosing IBD in an individual is particularly useful when the sensitivity is at least about 80%, or at least about 90%.
As used herein, the term "specificity" means the probability that a method is negative in the absence of IBD. Specificity is calculated as the number of true negative results divided by the sum of the true negatives and false positives. Specificity essentially is a measure of how well a method excludes those who do not have IBD. In a method of the invention, the ANCA, ASCA-IgA, and ASCA-IgG cut-off values "X," "Y," and "Z" can be selected such that, when the sensitivity is at least about 70%, the specificity of diagnosing an individual is in the range of 30-60%, for example, 35-60%, 40-60%, 45-60% or 50-60%. Furthermore, in a method of the invention, the ANCA, ASCA-IgA, and ASCA-IgG cut-off values "X," "Y," and "Z" can be selected such that, when the sensitivity is at least about 90%, the specificity of diagnosing an individual is in the range of 20-60%, for example, 20-30%, 20-40%, 20-50%, 30-60%, 35-60%, 40-60%, 45-60% or 50-60%. As illustrated in Example II, the maximum specificity that can be obtained in diagnosing IBD using a method of the invention is about 87.5%.
In a further embodiment, the ANCA, ASCA-IgA, and ASCA-IgG cut-off values "X," "Y," and "Z" can be independently selected, for example, such that the negative predictive value in a patient population having an IBD disease prevalence of about 15% is at least about 95%.
The term "negative predictive value," as used herein, is synonymous with "NPV" and means the probability that an individual diagnosed as not having IBD actually does not have the disease. Negative predictive value can be calculated as the number of true negatives divided by the sum of the true negatives and false negatives. Negative predictive value is determined by the characteristics of the diagnostic method as well as the prevalence of the disease in the population analyzed. In a method of the invention, the ANCA, ASCA-IgA, and ASCA-IgG cut-off values can be selected such that the negative predictive value in a population having an IBD disease prevalence is in the range of 80-99% and can be, for example, at least about 85%, at least about 90%, or at least about 95%. In particular, in a population having an IBD disease prevalence of 1 to 2%, the negative predictive value can be, for example, at least about 85%, 90%, 95%, 96%, 97%, 98% or 99%.
Predictive values, including negative and positive predictive values, are influenced by the prevalence of the disease in the population analyzed. In the methods of the invention, the cut-off values X, Y and Z can be selected to produce a desired clinical parameter for a clinical population with a particular IBD disease prevalence. For example, cut-off values X, Y and Z can be selected for an IBD disease prevalence of about 10%, 12%, 15%, 18% or 20%, which can be seen, for example, in a gastroenterologist's office. Cut-off values X, Y, and Z also can be selected for an IBD disease prevalence of about 1%, 2%, 3%, 4%, 5%, 6%, 7% or 8%. An IBD disease prevalence of 1 to 2% is typical of the disease prevalence seen in a general doctor's office.
In a method of the invention, the ANCA, ASCA-IgA, and ASCA-IgG cut-off values "X," "Y," and "Z" can be selected such that, when the sensitivity of diagnosing an individual with IBD is at least about 70% and the specificity of diagnosing an individual with IBD is in the range of 30-60%, the negative predictive value in a population having an IBD disease prevalence of about is at least about 90%. X, Y and Z can be selected such that, for example, the sensitivity is at least about 70%, the specificity is 30-60%, and the negative predictive value in a population having an IBD disease prevalence of about 15% is greater than 95%. Furthermore, in a method of the invention, the ANCA, ASCA-IgA, and ASCA-IgG cut-off values "X," "Y," and "Z" can be selected such that, when the sensitivity of diagnosing an individual with IBD is at least about 90% and the specificity of diagnosing an individual with IBD is in the range of 20-60%, the negative predictive value in a population having an IBD disease prevalence of about 15% is at least about 90%, for example, at least about 95%.
In a method of the invention, the ANCA, ASCA-IgA, and ASCA-IgG cut-off values "X," "Y," and "Z" can be selected such that, when the sensitivity of diagnosing an individual with IBD is at least about 70% and the specificity of diagnosing an individual with IBD is in the range of 20-60%, the negative predictive value in a population having an IBD disease prevalence of about 1-2% is at least about 98%. The values X, Y and Z can be selected such that the sensitivity is at least about 90%, the specificity of diagnosing an individual with IBD is 20-60%, and the negative predictive value in a population having an IBD disease prevalence of about 1-2% is greater than 98%. The negative predictive value in such a population can be, for example, greater than 99%.
In another embodiment, the ANCA, ASCA-IgA, and ASCA-IgG cut-off values "X," "Y," and "Z" can be independently selected such that, for example, the positive predictive value in a patient population having an IBD disease prevalence of about 15% is at least about 5%.
The term "positive predictive value," as used herein, is synonymous with "PPV" and means the probability that an individual diagnosed as having IBD actually has the disease. Positive predictive value can be calculated as the number of true positives divided by the sum of the true positives and false positives. Positive predictive value is determined by the characteristics of the diagnostic method as well as the prevalence of the disease in the population analyzed. In a method of the invention, the ANCA, ASCA-IgA, and ASCA-IgG cut-off values can be selected such that the positive predictive value of the method in a population having an IBD disease prevalence of 15% is at least about 5%, and can be, for example, at least about 8%, 10%, 15%, 20%, 25%, 30% or 40%.
In a further embodiment of the invention, the ANCA, ASCA-IgA, and ASCA-IgG cut-off values "X," "Y," and "Z" can be independently selected such that, for example, overall agreement in a patient population having an IBD disease prevalence of about 15% is at least about 45%.
As used herein, the term "overall agreement" means the accuracy with which a method diagnoses a disease state. Overall agreement is calculated as the sum of the true positives and true negatives divided by the total number of sample results and is affected by the prevalence of IBD in the population analyzed. The ANCA, ASCA-IgA, and ASCA-IgG cut-off values can be selected such that the overall agreement of a method of the invention in a patient population having an IBD disease prevalence of 15% is at least about 45%, and can be, for example, at least about 50%, 55% or 60%.
One skilled in the art can select an ANCA cut-off "X," an ASCA-IgA cut-off "Y," and an ASCA-IgG cut-off "Z" to achieve one or more clinically useful parameters, such as a desired sensitivity or specificity, or a desired negative predictive value, positive predictive value or overall agreement for a patient population having a particular disease prevalence. Factorial Design Optimization or Design of Experiments methodology can be used, for example, to select an appropriate ANCA cut-off "X," an ASCA-IgA cut-off "Y," and an ASCA-IgG cut-off "Z." As disclosed herein in Example II, optimization software (DOE Keep It Simple Statistically from Air Academy Associates (Colorado Springs, Colo.) was used in a central composite design experiment to simultaneously vary the three ELISA cut-offs "X," "Y," and "Z." In particular, the base ANCA cut-off was varied from 0.5 to 1.5 times the base value of approximately 10 to 20 EU; the base ASCA-IgA cut-off was varied from 10 EU to 30 EU; and the base ASCA-IgG cut-off was varied from 20 EU to 60 EU. By comparing the test results determined for the 851 individuals in the database (see Table 1) with the assigned "X," "Y," and "Z" cut-offs, each of the 851 samples were determined to be a true positive, true negative, false positive or false negative, and the clinical parameters of sensitivity, specificity, negative predictive value, positive predictive value and overall agreement were determined. Using these results, an optimized set of ANCA, ASCA-IgA and ASCA-IgG cut-off values were determined for each clinical parameter. Although the determination of the ANCA, ASCA-IgA and ASCA-IgG cut-off values "X," "Y," and "Z" is illustrated herein using the DOE KISS program, one skilled in the art understands that other computer programs for identifying cooperative interactions among multiple variables and for performing simultaneous equation calculations also can be used. For example, ECHIP optimization software, available from ECHIP, Incorporated (Hockessin, Del.), or Statgraphics optimization software, available from STSC, Incorporated (Rockville, Md.), also can be useful in determining the ANCA, ASCA-IgA and ASCA-IgG cut-off values in a method of the invention.
A variety of assay formats can be used to determine ANCA, ASCA-IgA and ASCA-IgG levels in a sample. As described above, the methods of the present invention can be performed with whole cells, such as neutrophils for the determination of ANCA levels, or yeast for the determination of ASCA-IgA or ASCA-IgG levels; with unpurified or partially purified cell extracts; or with purified proteins, protein fragments or peptides, which can be produced, for example, recombinantly, synthetically or using phage display technology.
Flow cytometry can be used to determine ANCA, ASCA-IgA and ASCA-IgG levels according to a method of the invention. Such flow cytometric assays, including bead based immunoassays, can be used to determine ANCA, ASCA-IgA and ASCA-IgG levels in the same manner as used to detect serum antibodies to Candida albicans and serum antibodies to HIV proteins (see, for example, Bishop and Davis, J. Immunol. Methods 210:79-87 (1997); McHugh et al., J. Immunol. Methods 116:213 (1989); Scillian et al., Blood 73:2041 (1989), each of which is incorporated by reference herein).
Phage display technology for expressing a recombinant antigen specific for ANCA or ASCA also can be used to determine the level of ANCA, ASCA-IgA or ASCA-IgG. Phage particles expressing the antigen specific for ANCA, or an antigen specific for ASCA, can be anchored, if desired, to a multiwell plate using an antibody such as an antiphage monoclonal antibody (Felici et al., "Phage-Displayed Peptides as Tools for Characterization of Human Sera" in Abelson (Ed.), Methods in Enzymol. 267, San Diego: Academic Press, Inc. (1996), which is incorporated by reference herein).
A variety of immunoassay formats including competitive and non-competitive immunoassay formats also are useful the methods of the invention (Self and Cook, Curr. Opin. Biotechnol. 7:60-65 (1996), which is incorporated by reference). Immunoassays encompass capillary electrophoresis based immunoassays (CEIA) and can be automated, if desired. Immunoassays also can be used in conjunction with laser induced fluorescence (see, for example, Schmalzing and Nashabeh, Electrophoresis 18:2184-93 (1997)); Bao, J. Chromatogr. B. Biomed. Sci. 699:463-80 (1997), each of which is incorporated herein by reference). Liposome immunoassays, such as flow-injection liposome immunoassays and liposome immunosensors, also can be used to determine ANCA, ASCA-IgA and ASCA-IgG levels according to a method of the invention (Rongen et al., J. Immunol. Methods 204:105-133 (1997), which is incorporated by reference herein).
Immunoassays, such as enzyme-linked immunosorbent assays (ELISAs), can be particularly useful in a method of the invention. A fixed neutrophil ELISA, for example, can be useful for determining whether a sample is positive for ANCA or for determining the ANCA level in a sample (see Example I). Similarly, an ELISA using yeast cell wall phosphopeptidomannan can be useful for determining whether a sample is positive for ASCA-IgA or ASCA-IgG, or for determining the ASCA-IgA or ASCA-IgG levels in a sample. An enzyme such as horseradish peroxidase (HRP), alkaline phosphatase (AP), β-galactosidase or urease can be linked to a secondary antibody selective for ANCA, or to a secondary antibody selective for ASCA for use in a method of the invention. A horseradish-peroxidase detection system can be used, for example, with the chromogenic substrate tetramethylbenzidine (TMB), which yields a soluble product in the presence of hydrogen peroxide that is detectable at 450 nm. An alkaline phosphatase detection system can be used with the chromogenic substrate p-nitrophenyl phosphate, for example, which yields a soluble product readily detectable at 405 nm. Similarly, a β-galactosidase detection system can be used with the chromogenic substrate o-nitrophenyl-β-D-galactopyranoside (ONPG), which yields a soluble product detectable at 410 nm, or a urease detection system can be used with a substrate such as urea-bromocresol purple (Sigma Immunochemicals, St. Louis, Mo.). A useful secondary antibody linked to an enzyme can be obtained from a number of commercial sources; goat F(ab')2 anti-human IgG-alkaline phosphatase, for example, can be purchased from Jackson Immuno-Research (West Grove, Pa.).
A radioimmunoassay also can be useful for determining whether a sample is positive for ANCA, ASCA-IgA or ASCA-IgG, or for determining the level of ANCA, ASCA-IgA or ASCA-IgG in a sample. A radioimmunoassay using, for example, an iodine-125 labeled secondary antibody (Harlow and Lane, Antibodies A Laboratory Manual Cold Spring Harbor Laboratory: New York, 1988, which is incorporated herein by reference) is encompassed within the invention.
A secondary antibody labeled with a chemiluminescent marker also can be useful in the methods of the invention. Such a chemiluminescent secondary antibody is convenient for sensitive, non-radioactive detection of ANCA, ASCA-IgA or ASCA-IgG and can be obtained commercially from various sources such as Amersham Lifesciences, Inc. (Arlington Heights, Ill.).
In addition, a detectable reagent labeled with a fluorochrome can be useful in the methods of the invention for determining whether ANCA, ASCA-IgA or ASCA-IgG is present in a sample. Appropriate fluorochromes include, for example, DAPI, fluorescein, Hoechst. 33258, R-phycocyanin, B-phycoerythrin, R-phycoerythrin, rhodamine, Texas red or lissamine. A particularly useful fluorochrome is fluorescein or rhodamine. Secondary antibodies linked to fluorochromes can be obtained commercially. For example, goat F(ab')2 anti-human IgG-FITC is available from Tago Immunologicals (Burlingame, Calif.).
A signal from the detectable reagent can be analyzed, for example, using a spectrophotometer to detect color from a chromogenic substrate; a radiation counter to detect radiation, such as a gamma counter for detection of iodine-125; or a fluorometer to detect fluorescence in the presence of light of a certain wavelength. For detection of enzyme-linked reagents, a quantitative analysis of the amount of ANCA, ASCA-IgA or ASCA-IgG can be made using a spectrophotometer such as an EMAX Microplate Reader (Molecular Devices, Menlo Park, Calif.) in accordance with the manufacturer's instructions. If desired, the assays of the invention can be automated or performed robotically, and the signal from multiple samples can be detected simultaneously.
Immunoassays using a secondary antibody selective for ANCA, or selective for ASCA-IgA, or selective for ASCA-IgG, are particularly useful in the methods of the invention. As used herein, the term "antibody" means a population of immunoglobulin molecules, which can be polyclonal or monoclonal and of any isotype. As used herein, the term "antibody" encompasses an immunologically active fragment of an immunoglobulin molecule. Such an immunologically active fragment contains the heavy and light chain variable regions, which make up the portion of the antibody molecule that specifically binds an antigen. For example, an immunologically active fragment of an immunoglobulin molecule known in the art as Fab, Fab' or F(ab')2 is included within the meaning of the term antibody.
The invention additionally provides a highly efficient method of analyzing multiple samples for IBD by first assaying all samples for the presence or absence of ANCA; next assaying only ANCA-negative samples for the presence or absence of ASCA-IgA; and next assaying only ANCA-negative and ASCA-IgA-negative samples for the presence or absence of ASCA-IgG, where the presence of pANCA, ASCA-IgA or ASCA-IgG in a sample is indicative of IBD and where the absence of ANCA, ASCA-IgA and ASCA-IgG is indicative of the absence of IBD. In such a method of the invention, the presence of ANCA, ASCA-IgA and ASCA-IgG can be conveniently determined, for example, using an immunoassay.
The following examples are intended to illustrate but not limit the present invention.
PAC Determination of Patient ANCA StatusThis example describes anlysis of patient ANCA, ASCA-IgA and ASCA-IgG levels using ELISA assays.
A. Fixed Neutrophil ELISA for Determining ANCA Levels
A fixed neutrophil enzyme-linked immunosorbent assay was used to detect ANCA as described in Saxon et al., supra, 1990. Briefly, microtiter plates were coated with 2.5×105 neutrophils per well from peripheral human blood purified by Ficoll-hypaque centrifugation and treated with 100% methanol for 10 minutes to fix the cells. Cells were incubated with 0.25% bovine serum albumin (BSA) in phosphate-buffered saline to block nonspecific antibody binding for 60 minutes at room temperature in a humidified chamber. Next, control and coded sera were added at a 1:100 dilution to the bovine serum/phosphate-buffered saline blocking buffer and incubated for 60 minutes at room temperature in a humidified chamber. Alkaline phosphatase-conjugated goat F(ab')2 anti-human immunoglobulin G antibody (γ-chain specific; Jackson Immunoresearch Labs, Inc., West Grove, Pa.) was added at a 1:1000 dilution to label neutrophil-bound antibody and incubated for 60 minutes at room temperature. A solution of p-nitrophenol phosphate substrate was added, and color development was allowed to proceed until absorbance at 405 nm in the positive control wells was 0.8-1.0 optical density units greater than the absorbance in blank wells.
A panel of twenty verified negative control samples was used with a calibrator with a defined ELISA Unit (EU) value. The base positive/negative cut-off for each ELISA run was defined as the optical density (OD) of the Calibrator minus the mean (OD) value for the panel of twenty negatives (plus 2 standard deviations) times the EU value of the Calibrator. The base cut-off value for ANCA reactivity was therefore about 10 to 20 EU, with any patient sample having an average EU value greater than the base cut-off marked as ELISA positive for ANCA reactivity. Similarly, a patient sample having an average EU value is less than or equal to the base cut-off is determined to be negative for ANCA reactivity.
B. Preparation of Yeast Cell Wall Mannan for ASCA ELISA Assay
Yeast cell wall mannan was prepared as follows and as described in Faille et al., Eur. J. Clin. Microbiol. Infect. Dis. 11:438-446 (1992) and in Kocourek and Ballou et al., J. Bacteriol 100:1175-1181 (1969), each of which is incorporated herein by reference. A lyophilized pellet of yeast Saccharomyces uvarum was obtained from the American Type Culture Collection (#38926). Yeast were reconstituted in 10 ml 2× YT medium, prepared according to Sambrook et al., Molecular Cloning Cold Spring Harbor Laboratory Press (1989), which is incorporated herein by reference. S. uvarum were grown for two to three days at 30°C The terminal S. uvarum culture was inoculated on a 2× YT agar plate and subsequently grown for two to three days at 30°C A single colony was used to inoculate 500 ml 2× YT media, and grown for two to three days at 30°C Fermentation media (pH 4.5) was prepared by adding 20 gm glucose, 2 gm bacto-yeast extract, 0.25 gm MgSO4 and 2.0 ml 28% H3 PO4 per liter distilled water. The 500 ml culture was used to inoculate 50 liters of fermentation media, and the culture fermented for three to four days at 37°C
S. uvarum mannan extract was prepared by adding 50 ml 0.02 M citrate buffer (5.88 gm/l sodium citrate; pH 7.0+/-0.1) to each 100 grams of cell paste. The cell/citrate mixture was autoclaved at 125°C for ninety minutes and allowed to cool. After centrifuging at 5000 rpm for 10 minutes, the supernatant was removed and retained. The cells were then washed with 75 ml 0.02 M citrate buffer and the cell/citrate mixture again autoclaved at 125°C for ninety minutes. The cell/citrate mixture was centrifuged at 5000 rpm for 10 minutes, and the supernatant retained.
In order to precipitate copper/mannan complexes, an equal volume of Fehling's Solution was added to the combined supernatants while stirring. The complete Fehling's solution was prepared by mixing Fehling's Solution A with Fehling's Solution B in a 1:1 ratio just prior to use. The copper complexes were allowed to settle, and the liquid decanted gently from the precipitate. The copper/mannan precipitate complexes were then dissolved in 6-8 ml 3N HCl per 100 grams yeast paste.
The resulting solution was poured with vigorous stirring into 100 ml of 8:1 methanol:acetic acid, and the precipitate allowed to settle for several hours. The supernatant was decanted and discarded; then the wash procedure was repeated until the supernatant was colorless, approximately two to three times. The precipitate was collected on a scintered glass funnel, washed with methanol and air dried overnight. On some occasions, the precipitate was collected by centrifugation at 5000 rpm for 10 minutes before washing with methanol and air drying overnight. The dried mannan ;powder was dissolved in distilled water, to a concentration of approximately 2 g/ml.
C. Preparation of S. uvarum Mannan ELISA Plates S. uvarum cell mannan ELISA plates were saturated with antigen as follows. Purified S. uvarum mannan prepared as described above was diluted to a concentration of 100 μg/ml with phosphate buffered saline/0.2% sodium azide (PBS-N3). Using a multi-channel pipettor, 100 μl of 100 μg/ml S. uvarum mannan was added per well of a Costar 96-well hi-binding plate (catalogue number 3590; Costar Corp., Cambridge, Mass.). The antigen was allowed to coat the plate at 4°C for a minimum of 12 hours. Each lot of plates was compared to a previous lot before use. Plates were stored at 2-8° C. for up to one month.
D. ASCA ELISA Analysis of Patient Sera
Patient sera were analyzed in duplicate for anti-IgG or anti-IgA reactivity. Microtiter plates saturated with antigen as described above were incubated with-phosphate buffered saline/0.05% Tween-20 for 45 minutes at room temperature to inhibit nonspecific antibody binding. Patient sera were subsequently added at a dilution of 1:80 for ASCA-IgA and 1:800 for analysis of ASCA-IgG and incubated for 1 hour at room temperature. Wells were washed three times with PBS/0.05% Tween-20. Then a 1:1000 dilution of alkaline phosphatase-conjugated goat anti-human IgA (Jackson Immunoresearch, West Grove, Pa.) or a 1:1000 dilution of alkaline phosphatase-conjugated goat anti-human IgG F(ab')2 (Pierce, Rockford, Ill.) or was added, and the microtiter plates incubated for 1 hour at room temperature. A solution of p-nitrophenol phosphate in diethanolamine substrate buffer was added, and color development allowed to proceed for 10 minutes. Absorbance at 405 nm was analyzed using an automated EMAX plate reader (Molecular Devices, Sunnyvale, Calif.).
To determine the base cut-off value for ASCA-IgA and ASCA-IgG, single point calibrators having fixed EU values were used. OD values for patients samples were compared to the OD value for the Calibrators and multiplied by the Calibrator assigned values. The base cut-off value for the ASCA-IgA ELISA was 20 EU. The base cut-off value for the ASCA-IgG was 40 EU.
PAC Determination of Optimized Cut-Offs for ANCA, ASCA-IgA and ASCA-IgG PositivityThis example demonstrates that particular ANCA, ASCA-IgA and ASCA-IgG cut-off values can be selected to yield a preferred clinical parameter for diagnosing IBD.
A. Database Used in Analysis
Only quantitative ELISA procedures were performed and particular cut-off values for the results of each ELISA assay used to determine whether the test serum sample was positive or negative for markers of IBD. In particular, no immunofluorescence assay procedures were performed as part of these diagnostics.
The cut-off values for each of the three ELISA components of the assay were determined using a database consisting of serology data from 851 individuals (Table 1). The presence or absence of inflammatory bowel disease was made for all IBD patients by colonoscopic, radiologic, and/or histologic methods at Cedars Sinai Medical Center (Los Angeles, Calif.). Serum from 300 asymptomatic non-disease controls also was tested, although, for ethical reasons, colonoscopy was not performed on these individuals.
| TABLE 1 |
| ANCA, ASCA-IgA and ASCA-IgG serology data from 851 person database |
| ANCA ANCA ASCA-IgA |
| ASCA-IgG |
| SAMPLE IBD ELISA ELISA RESULT |
| RESULT |
| COUNT ID DIAGNOSIS CLASS RESULT CUT-OFF (EU) |
| (EU) |
| 1 1 NON IBD NON IBD 8.624 13.374 5.356 |
| 15.516 |
| 2 2 NON IBD NON IBD 6.606 13.374 1.379 |
| 10.28 |
| 3 3 NON IBD NON IBD 6.056 13.374 3.995 |
| 5.874 |
| 4 4 NON IBD NON IBD 9.602 13.374 1.697 |
| 12.77 |
| 5 5 NON IBD NON IBD 6.85 13.374 1.626 |
| 6.449 |
| 6 6 NON IBD NON IBD 7.462 13.374 1.997 |
| 12.898 |
| 7 7 NON IBD NON IBD 7.278 13.374 2.669 |
| 8.684 |
| 8 8 NON IBD NON IBD 15.229 13.374 2.881 |
| 11.429 |
| 9 9 NON IBD NON IBD 11.743 13.374 1.591 |
| 17.431 |
| 10 10 NON IBD NON IBD 13.7 13.374 0.389 |
| 4.533 |
| 11 11 NON IBD NON IBD 12.783 13.374 2.492 |
| 23.434 |
| 12 12 NON IBD NON IBD 11.07 13.374 3.853 |
| 6.96 |
| 13 13 NON IBD NON IBD 9.297 13.374 11.754 |
| 10.599 |
| 14 14 NON IBD NON IBD 7.951 13.374 0.548 |
| 4.533 |
| 15 15 NON IBD NON IBD 9.833 11.989 0.716 |
| 6.065 |
| 16 16 NON IBD NON IBD 8.135 13.374 0.813 |
| 5.938 |
| 17 17 NON IBD NON IBD 7.829 13.374 12.39 |
| 7.471 |
| 18 18 NON IBD NON IBD 11.988 13.374 2.863 |
| 26.371 |
| 19 19 NON IBD NON IBD 5.015 13.374 1.891 |
| 5.938 |
| 20 20 NON IBD NON IBD 9.358 13.374 1.98 |
| 4.278 |
| 21 21 NON IBD NON IBD 8.073 13.374 1.573 |
| 5.747 |
| 22 22 NON IBD NON IBD 9.419 13.374 2.492 |
| 11.94 |
| 23 23 NON IBD NON IBD 5.015 13.374 1.962 |
| 6.641 |
| 24 24 NON IBD NON IBD 7.278 13.374 1.502 |
| 13.537 |
| 25 25 NON IBD NON IBD 9.419 13.374 3.252 |
| 8.875 |
| 26 26 NON IBD NON IBD 9.174 13.374 0.442 |
| 4.883 |
| 27 27 NON IBD NON IBD 19.388 13.374 5.285 |
| 7.854 |
| 28 28 NON IBD NON IBD 12.355 13.374 2.563 |
| 5.108 |
| 29 29 NON IBD NON IBD 6.544 13.374 5.638 |
| 5.172 |
| 30 30 NON IBD NON IBD 7.278 13.374 13.504 |
| 15.58 |
| 31 31 NON IBD NON IBD 7.645 13.374 4.277 |
| 27.137 |
| 32 32 NON IBD NON IBD 8.624 13.374 2.722 |
| 4.661 |
| 33 33 NON IBD NON IBD 20.183 13.374 3.411 |
| 6.002 |
| 34 34 NON IBD NON IBD 14.434 13.374 15.006 |
| 19.794 |
| 35 35 NON IBD NON IBD 16.566 20.956 6.345 |
| 12.643 |
| 36 36 NON IBD NON IBD 17.9 20.956 13.398 |
| 17.559 |
| 37 37 NON IBD NON IBD 10.801 20.956 2.474 |
| 5.683 |
| 38 38 NON IBD NON IBD 23.726 20.956 22.642 |
| 37.034 |
| 39 39 NON IBD NON IBD 16.323 20.956 1.803 |
| 18.198 |
| 40 40 NON IBD NON IBD 19.114 20.956 4.313 |
| 11.429 |
| 41 41 NON IBD NON IBD 22.33 20.956 3.888 |
| 5.108 |
| 42 42 NON IBD NON IBD 6.371 20.956 2.404 |
| 2.554 |
| 43 43 NON IBD NON IBD 17.597 20.956 13.804 |
| 19.475 |
| 44 44 NON IBD NON IBD 14.745 20.956 1.326 |
| 2.937 |
| 45 45 NON IBD NON IBD 7.16 20.956 1.827 |
| 4.373 |
| 46 46 NON IBD NON IBD 13.896 20.956 1.292 |
| 2.345 |
| 47 47 NON IBD NON IBD 7.585 20.956 1.846 |
| 6.782 |
| 48 48 NON IBD NON IBD 10.437 20.956 0.941 |
| 5.134 |
| 49 49 NON IBD NON IBD 7.282 20.956 1.034 |
| 21.296 |
| 50 50 NON IBD NON IBD 13.774 20.956 1.569 |
| 7.606 |
| 51 51 NON IBD NON IBD 17.718 20.956 2.215 |
| 6.021 |
| 52 52 NON IBD NON IBD 11.408 20.956 0.646 |
| 2.535 |
| 53 53 NON IBD NON IBD 7.949 20.956 2.363 |
| 9.571 |
| 54 54 NON IBD NON IBD 14.684 20.956 1.218 |
| 5.704 |
| 55 55 NON IBD NON IBD 14.867 20.956 3.784 |
| 7.986 |
| 56 56 NON 1Bb NON IBD 11.529 20.956 1.2 |
| 10.775 |
| 57 57 NON IBD NON IBD 21.177 20.956 2.196 |
| 8.43 |
| 58 58 NON IBD NON IBD 9.527 20.956 2.639 |
| 9.697 |
| 59 59 NON IBD NON IBD 12.985 20.956 2.16 |
| 5.894 |
| 60 60 NON IBD NON IBD 11.59 20.956 2.436 |
| 22.881 |
| 61 61 NON IBD NON IBD 10.255 20.956 0.554 |
| 2.028 |
| 62 62 NON IBD NON IBD 13.592 20.956 1.218 |
| 5.958 |
| 63 63 NON IBD NON IBD 13.532 20.956 1.347 |
| 15.211 |
| 64 64 NON IBD NON IBD 12.500 20.956 11.228 |
| 98.739 |
| 65 65 NON IBD NON IBD 12.925 20.956 2.170 |
| 12.131 |
| 66 66 NON IBD NON IBD 12.318 20.956 3.780 |
| 12.907 |
| 67 67 NON IBD NON IBD 16.262 20.956 7.717 |
| 18.478 |
| 68 68 NON IBD NON IBD 8.897 21.656 1.767 |
| 3.667 |
| 69 69 NON IBD NON IBD 7.374 21.656 1.767 |
| 6.7 |
| 70 70 NON IBD NON IBD 17.550 21.656 0.895 |
| 33.501 |
| 71 71 NON IBD NON IBD 20.963 21.656 6.554 |
| 14.247 |
| 72 72 NON IBD NON IBD 36.868 21.656 2.774 |
| 7.264 |
| 73 73 NON IBD NON IBD 15.113 21.656 1.566 |
| 12.342 |
| 74 74 NON IBD NON IBD 12.371 21.656 12.749 |
| 9.521 |
| 75 75 NON IBD NON IBD 13.163 21.656 2.505 |
| 4.373 |
| 76 76 NON IBD NON IBD 20.353 21.656 2.505 |
| 8.745 |
| 77 77 NON IBD NON IBD 28.032 21.656 4.317 |
| 28.211 |
| 78 78 NON IBD NON IBD 14.869 21.656 1.253 |
| 4.866 |
| 79 79 NON IBD NON IBD 15.174 21.656 4.719 |
| 10.297 |
| 80 80 NON IBD NON IBD 18.952 21.656 5.681 |
| 32.937 |
| 81 81 NON IBD NON IBD 25.960 21.656 36.347 |
| 43.868 |
| 82 82 NON IBD NON IBD 18.342 21.656 2.438 |
| 11.426 |
| 83 83 NON IBD NON IBD 21.511 21.656 1.096 |
| 5.36 |
| 84 84 NON IBD NON IBD 17.002 21.656 1.856 |
| 6.841 |
| 85 85 NON IBD NON IBD 18.282 21.656 4.004 |
| 7.758 |
| 86 86 NON IBD NON IBD 8.775 21.656 2.572 |
| 2.116 |
| 87 87 NON IBD NON IBD 12.066 21.656 4.742 |
| 8.534 |
| 88 88 NON IBD NON IBD 15.844 21.656 4.563 |
| 29.974 |
| 89 89 NON IBD NON IBD 15.722 21.656 1.230 |
| 2.892 |
| 90 90 NON IBD NON IBD 13.528 21.656 1.163 |
| 4.373 |
| 91 91 NON IBD NON IBD 19.988 21.656 6.129 |
| 9.169 |
| 92 92 NON IBD NON IBD 17.002 21.656 4.317 |
| 25.672 |
| 93 93 NON IBD NON IBD 18.647 21.656 0.962 |
| 6.982 |
| 94 94 NON IBD NON IBD 17.733 21.656 15.545 |
| 33.994 |
| 95 95 NON IBD NON IBD 16.819 21.656 9.327 |
| 16.715 |
| 96 96 NON IBD NON IBD 17.550 21.656 7.269 |
| 18.196 |
| 97 97 NON IBD NON IBD 13.833 21.656 3.422 |
| 9.733 |
| 98 98 NON IBD NON IBD 7.861 21.656 10.244 |
| 7.123 |
| 99 99 NON IBD NON IBD 14.747 21.656 2.930 |
| 13.048 |
| 100 100 NON IBD NON IBD 15.661 21.656 3.623 |
| 15.869 |
| 101 101 NON IBD NON IBD 15.697 11.989 3.646 |
| 13.4 |
| 102 102 NON IBD NON IBD 11.231 11.989 2.214 |
| 10.156 |
| 103 103 CD IBD 10.311 17.023 11.264 |
| 14.32 |
| 104 104 CD IBD 49.604 17.023 4.613 |
| 45.98 |
| 105 107 CD IBD 12.919 17.023 130.938 |
| 62.82 |
| 106 108 CD IBD 21.242 17.023 115.841 |
| 85.92 |
| 107 110 CD IBD 35.031 17.023 61.667 |
| 57.82 |
| 108 111 UC IBD 6.708 17.023 31.708 |
| 36.87 |
| 109 113 UC IBD 16.149 17.023 18.369 |
| 12.54 |
| 110 114 CD IBD 19.565 17.023 22.501 |
| 31.03 |
| 111 115 CD IBD 23.168 17.023 45.229 |
| 33.71 |
| 112 116 CD IBD 16.335 17.023 10.974 |
| 7.46 |
| 113 118 UC IBD 24.161 17.023 20.816 |
| 49.51 |
| 114 119 UC IBD 74.596 17.023 8.419 |
| 13.82 |
| 115 122 UC IBD 150.215 17.023 4.178 |
| 7.80 |
| 116 126 CD IBD 17.019 17.023 128.111 |
| 84.87 |
| 117 127 CD IBD 28.509 17.023 42.546 |
| 40.00 |
| 118 134 CD IBD 30.932 17.023 9.071 |
| 8.27 |
| 119 136 UC IBD 51.977 17.023 67.842 |
| 16.81 |
| 120 137 UC IBD 98.417 17.023 4.558 |
| 13.78 |
| 121 138 CONTROL NON IBD 14.845 17.023 2.492 |
| 13.88 |
| 122 139 CD IBD 9.503 17.023 5.682 |
| 8.57 |
| 123 140 CD IBD 49.683 17.023 18.840 |
| 10.66 |
| 124 142 UC IBD 118.275 17.023 1.912 |
| 4.30 |
| 125 143 CD IBD 22.919 17.023 4.993 |
| 11.66 |
| 126 144 CD IBD 18.820 17.023 55.270 |
| 27.43 |
| 127 145 CD IBD 29.130 17.023 137.028 |
| 118.39 |
| 128 146 CONTROL NON IBD 38.199 17.023 18.677 |
| 19.53 |
| 129 147 UC IBD 58.306 17.023 2.347 |
| 5.71 |
| 130 148 CD IBD 44.472 17.023 41.658 |
| 25.58 |
| 131 149 CD IBD 65.189 17.023 41.260 |
| 14.55 |
| 132 157 CD IBD 17.888 17.023 27.431 |
| 24.61 |
| 133 158 CD IBD 54.836 17.023 61.831 |
| 24.91 |
| 134 160 UC IBD 162.181 17.023 8.292 |
| 9.82 |
| 135 166 CD IBD 27.578 17.023 36.964 |
| 22.86 |
| 136 168 CD IBD 21.615 17.023 134.527 |
| 160.93 |
| 137 169 UC IBD 88.000 17.023 9.434 |
| 8.15 |
| 138 170 CD IBD 32.298 17.023 71.201 |
| 48.56 |
| 139 174 UC IBD 56.072 17.023 10.720 |
| 15.04 |
| 140 175 CD IBD 43.416 17.023 2.691 |
| 14.38 |
| 141 176 NON IBD NON IBD 14.472 17.023 6.588 |
| 5.68 |
| 142 178 CD IBD 43.054 17.023 80.807 |
| 64.06 |
| 143 201 CD IBD 16.211 17.023 31.980 |
| 35.14 |
| 144 203 CD IBD 18.758 17.023 73.394 |
| 35.85 |
| 145 204 CD IBD 58.400 17.023 57.010 |
| 112.83 |
| 146 206 CD IBD 10.870 17.023 47.386 |
| 34.81 |
| 147 207 CD IBD 11.304 17.023 95.421 |
| 60.64 |
| 148 213 CD IBD 34.721 17.023 39.549 |
| 15.55 |
| 149 214 CD IBD 20.311 17.023 27.289 |
| 25.37 |
| 150 216 CD IBD 43.272 17.023 57.880 |
| 33.85 |
| 151 218 CD IBD 21.677 17.023 23.242 |
| 36.56 |
| 152 221 NON IBD NON IBD 15.776 17.023 22.296 |
| 10.74 |
| 153 223 CONTROL NON IBD 8.025 15.673 16.938 |
| 9.44 |
| 154 224 CD IBD 14.502 15.673 27.256 |
| 52.74 |
| 155 229 CD IBD 13.474 15.673 8.129 |
| 14.92 |
| 156 231 CD IBD 50.254 15.673 97.014 |
| 69.24 |
| 157 234 CD IBD 46.690 15.673 45.405 |
| 36.68 |
| 158 236 UC IBD 32.568 15.673 7.150 |
| 41.93 |
| 159 237 UC IBD 70.695 15.673 7.780 |
| 8.56 |
| 160 238 CONTROL NON IBD 11.239 15.673 2.820 |
| 10.32 |
| 161 247 UC IBD 24.109 15.673 25.747 |
| 32.92 |
| 162 248 CD IBD 19.517 15.673 14.615 |
| 21.30 |
| 163 249 CD IBD 5.740 15.673 3.368 |
| 5.89 |
| 164 253 CD IBD 30.453 15.673 7.697 |
| 17.54 |
| 165 254 UC IBD 70.028 15.673 4.745 |
| 15.50 |
| 166 258 NON IBD NON IBD 11.904 15.673 4.197 |
| 10.59 |
| 167 260 CD IBD 59.238 15.673 7.100 |
| 44.96 |
| 168 261 NON IBD NON IBD 41.390 15.673 3.019 |
| 9.98 |
| 169 262 CD IBD 7.855 15.673 2.273 |
| 11.56 |
| 170 265 UC IBD 73.131 15.673 4.214 |
| 10.80 |
| 171 267 CD IBD 20.181 15.673 72.512 |
| 23.45 |
| 172 274 UC IBD 139.985 15.673 14.267 |
| 11.47 |
| 173 275 CD IBD 25.921 15.673 10.186 |
| 11.62 |
| 174 276 UC IBD 93.582 15.673 11.347 |
| 18.33 |
| 175 278 CD IBD 94.005 15.673 86.447 |
| 97.21 |
| 176 314 UC IBD 15.529 15.673 8.245 |
| 15.56 |
| 177 321 UC IBD 157.281 15.673 11.032 |
| 16.35 |
| 178 325 NON IBD NON IBD 13.716 15.673 10.687 |
| 27.15 |
| 179 329 CD IBD 8.097 15.673 3.878 |
| 7.68 |
| 180 334 IBS NON IBD 54.583 15.673 3.557 |
| 26.79 |
| 181 339 UC IBD 55.589 15.673 3.846 |
| 9.28 |
| 182 352 CD IBD 24.592 15.673 124.983 |
| 129.22 |
| 183 362 UC IBD 17.100 15.673 1.698 |
| 5.49 |
| 184 374 NON IBD NON IBD 9.305 15.673 6.885 |
| 13.80 |
| 185 376 CD IBD 13.172 15.673 119.436 |
| 18.33 |
| 186 377 NON IBD NON IBD 12.508 15.673 18.331 |
| 8.83 |
| 187 384 UC IBD 65.257 15.673 4.529 |
| 6.64 |
| 188 403 UC IBD 83.950 15.673 18.613 |
| 36.49 |
| 189 405 UC IBD 89.737 15.673 13.869 |
| 27.63 |
| 190 406 CONTROL NON IBD 19.795 15.673 5.889 |
| 12.40 |
| 191 407 CD IBD 6.949 15.673 4.960 |
| 29.41 |
| 192 409 CD IBD 114.898 15.673 18.547 |
| 26.60 |
| 193 413 UC IBD 24.900 15.673 2.107 |
| 9.60 |
| 194 413 UC IBD 24.894 15.673 1.955 |
| 9.6 |
| 195 414 CD IBD 54.475 15.673 67.817 |
| 102.23 |
| 196 416 CONTROL NON IBD 15.693 15.673 6.437 |
| 31.19 |
| 197 417 UC IBD 133.897 15.673 4.147 |
| 19.22 |
| 198 419 CD IBD 4.411 15.673 27.090 |
| 79.19 |
| 199 420 CD IBD 23.021 15.673 5.410 |
| 32.59 |
| 200 421 CD IBD 65.500 15.673 43.334 |
| 30.13 |
| 201 421 CD IBD 65.509 15.673 23.073 |
| 30.13 |
| 202 422 CONTROL NON IBD 22.425 15.673 8.714 |
| 29.56 |
| 203 423 UC IBD 37.523 15.673 7.942 |
| 10.85 |
| 204 424 CD IBD 18.429 15.673 33.448 |
| 32.74 |
| 205 425 IBS NON IBD 6.100 15.673 0.813 |
| 3.35 |
| 206 425 NON IBD NON IBD 6.103 15.673 0.625 |
| 3.35 |
| 207 426 NON IBD NON IBD 14.743 15.673 4.150 |
| 4.90 |
| 208 428 CD IBD 57.780 15.673 7.583 |
| 9.45 |
| 209 429 CD IBD 36.469 15.673 6.894 |
| 9.18 |
| 210 434 NON IBD NON IBD 16.073 15.673 -1.351 |
| 16.11 |
| 211 437 IBS NON IBD 16.435 15.673 5.763 |
| 17.40 |
| 212 438 CD IBD 64.824 15.673 60.141 |
| 36.22 |
| 213 443 IBS NON IBD 10.808 15.673 -0.262 6.12 |
| 214 444 CD IBD 26.326 15.673 27.134 |
| 42.89 |
| 215 449 CONTROL NON IBD 28.266 15.673 16.531 |
| 36.49 |
| 216 450 UC IBD 29.598 15.673 3.157 |
| 18.20 |
| 217 451 IBS NON IBD 7.238 15.673 -2.344 5.81 |
| 218 453 UC/PSC IBD 58.404 15.673 7.555 |
| 12.67 |
| 219 455 UC IBD 65.354 15.673 11.375 |
| 16.34 |
| 220 458 CONTROL NON IBD 38.163 15.673 3.819 |
| 8.54 |
| 221 461 UC IBD 50.694 15.673 28.292 |
| 14.90 |
| 222 464 CD IBD 30.094 15.673 100.041 |
| 126.70 |
| 223 502 CD IBD 6.247 15.673 8.727 |
| 26.30 |
| 224 504 NON IBD NON IBD 14.080 15.673 0.345 |
| 16.80 |
| 225 505 NON IBD NON IBD 9.321 15.673 5.418 |
| 20.21 |
| 226 506 CONTROL NON IBD 23.451 15.673 12.643 |
| 36.35 |
| 227 509 CD IBD 64.285 15.673 10.258 |
| 25.51 |
| 228 515 CONTROL NON IBD 17.744 15.673 8.934 |
| 23.81 |
| 229 531 CD IBD 198.079 15.673 4.829 |
| 20.58 |
| 230 534 CONTROL NON IBD 30.049 15.673 12.241 |
| 43.53 |
| 231 536 IBS NON IBD 67.281 54.120 14.554 |
| 34.46 |
| 232 537 NON IBD NON IBD 22.607 24.358 1.988 |
| 21.49 |
| 233 538 CD IBD 65.898 54.120 98.850 |
| 174.86 |
| 234 539 CD IBD 14.477 24.358 3.463 |
| 22.89 |
| 235 542 CONTROL NON IBD 11.057 15.673 4.234 |
| 30.99 |
| 236 543 UC IBD 69.278 54.120 7.521 |
| 21.74 |
| 237 544 CD IBD 15.915 24.358 27.458 |
| 57.35 |
| 238 545 NON IBD NON IBD 18.245 24.358 53.402 |
| 90.17 |
| 239 547 IBS NON IBD 9.817 24.358 13.080 |
| 34.58 |
| 240 551 IBS NON IBD 12.643 24.358 4.220 |
| 36.53 |
| 241 552 CD IBD 71.812 54.120 16.313 |
| 43.47 |
| 242 553 CD IBD 30.788 24.358 11.335 |
| 69.35 |
| 243 554 CD IBD 17.501 24.358 80.752 |
| 97.96 |
| 244 557 NON IBD NON IBD 11.701 24.358 5.248 |
| 24.11 |
| 245 559 UC IBD 90.564 69.938 5.140 |
| 28.25 |
| 246 560 IBS NON IBD 17.253 24.358 5.343 |
| 61.37 |
| 247 561 UC IBD 17.005 24.358 20.506 |
| 22.53 |
| 248 562 UC IBD 23.688 20.189 38.929 |
| 26.18 |
| 249 563 CD IBD 26.338 20.189 3.076 |
| 47.37 |
| 250 566 CONTROL NON IBD 18.601 20.189 5.613 |
| 32.88 |
| 251 567 UC IBD 79.012 69.938 6.154 |
| 11.29 |
| 252 569 CD IBD 19.873 20.189 -0.054 |
| 26.60 |
| 253 570 UC IBD 111.640 20.189 14.351 |
| 19.49 |
| 254 574 IBS NON IBD 20.138 20.189 2.448 |
| 13.77 |
| 255 577 IBS NON IBD 13.620 20.189 0.460 |
| 2.68 |
| 256 578 UC IBD 21.516 20.189 0.852 |
| 4.78 |
| 257 579 UC IBD 93.827 69.938 -0.257 5.64 |
| 258 580 CD IBD 18.283 20.189 23.874 |
| 16.22 |
| 259 581 CD IBD 18.177 20.189 61.923 |
| 48.26 |
| 260 601 CD IBD 20.880 20.189 1.772 |
| 6.20 |
| 261 603 UC IBD 78.571 69.938 4.153 |
| 6.20 |
| 262 604 UC IBD 189.947 69.938 7.291 |
| 27.68 |
| 263 605 UC IBD 37.149 20.189 3.896 |
| 9.00 |
| 264 608 UC IBD 76.984 69.934 29.533 |
| 23.72 |
| 265 609 UC/PSC IBD 60.934 69.934 22.143 |
| 24.90 |
| 266 610 UC IBD 67.356 20.189 4.634 |
| 3.63 |
| 267 613 UC IBD 50.026 20.189 5.018 |
| 22.93 |
| 268 616 UC IBD 86.507 69.938 5.519 |
| 12.9 |
| 269 622 UC IBD 50.609 20.189 9.327 |
| 13.93 |
| 270 623 UC IBD 117.724 69.938 6.198 |
| 8.48 |
| 271 625 CD IBD 13.937 20.189 27.597 |
| 39.58 |
| 272 627 NON IBD NON IBD 13.620 20.189 2.420 |
| 6.24 |
| 273 628 CD IBD 77.865 69.938 131.730 |
| 225.41 |
| 274 629 UC/PSC IBD 27.663 20.189 2.656 |
| 4.62 |
| 275 631 IBS NON IBD 14.467 20.189 5.224 |
| 10.54 |
| 276 632 UC IBD 86.963 20.189 6.523 |
| 12.43 |
| 277 633 NON IBD NON IBD 18.972 20.189 1.623 |
| 3.47 |
| 278 634 IBS NON IBD 17.064 20.189 3.040 |
| 6.55 |
| 279 637 UC IBD 154.637 20.189 4.162 |
| 3.51 |
| 280 639 UC IBD 85.957 20.189 6.612 |
| 18 |
| 281 647 NON IBD NON IBD 14.891 20.189 2.066 |
| 6.08 |
| 282 648 UC IBD 63.328 20.189 4.309 |
| 110.18 |
| 283 650 CD IBD 38.951 20.189 157.231 |
| 92.74 |
| 284 651 UC IBD 59.088 20.189 47.816 |
| 34.93 |
| 285 660 UC IBD 85.841 16.705 5.106 |
| 17.09 |
| 286 661 NON IBD NON IBD 12.049 16.705 2.273 |
| 7.56 |
| 287 663 CD IBD 9.606 16.705 89.138 |
| 61.33 |
| 288 667 UC IBD 27.762 16.705 4.723 |
| 10.12 |
| 289 668 UC IBD 9.328 16.705 2.509 |
| 8.84 |
| 290 669 CD IBD 14.881 16.705 1.860 |
| 11.31 |
| 291 672 UC IBD 116.380 16.705 4.634 |
| 19.16 |
| 292 678 CD IBD 9.717 16.705 2.037 |
| 17.43 |
| 293 679 CD IBD 78.956 16.705 0.974 |
| 9.78 |
| 294 681 UC IBD 8.329 16.705 1.771 |
| 16.25 |
| 295 702 CD IBD 6.441 16.705 1.033 |
| 2.57 |
| 296 703 CD IBD 18.656 16.705 87.426 |
| 81.04 |
| 297 704 UC IBD 33.981 16.705 11.959 |
| 24.79 |
| 298 705 CD IBD 35.702 16.705 16.102 |
| 23.65 |
| 299 706 UC IBD 10.161 16.705 122.505 |
| 87.7 |
| 300 707 UC IBD 12.993 16.705 4.175 |
| 18.02 |
| 301 709 CD IBD 42.754 16.705 60.923 |
| 71.46 |
| 302 711 IBS NON IBD 17.990 16.705 7.094 |
| 15.51 |
| 303 712 CD IBD 19.267 16.705 38.449 |
| 38.57 |
| 304 714 CD IBD 74.014 16.705 1.820 |
| 3.85 |
| 305 716 UC IBD 127.263 16.705 5.618 |
| 12 |
| 306 717 CD IBD 11.105 16.705 62.398 |
| 37.83 |
| 307 718 UC IBD 34.703 16.705 5.085 |
| 33.09 |
| 308 719 CD IBD 23.320 16.705 5.304 |
| 12.89 |
| 309 720 CD IBD 38.701 16.705 165.851 |
| 115.85 |
| 310 721 UC IBD 101.444 16.705 1.507 |
| 4.64 |
| 311 725 IBS NON IBD 16.687 23.307 3.296 |
| 8.99 |
| 312 727 CD IBD 16.872 23.307 2.103 |
| 22.75 |
| 313 728 CD IBD 36.761 23.307 8.600 |
| 8.29 |
| 314 729 UC IBD 94.828 23.307 2.103 |
| 7.48 |
| 315 730 CD IBD 15.517 23.307 12.084 |
| 10.52 |
| 316 732 UC IBD 18.350 23.307 1.507 |
| 7.33 |
| 317 733 CD IBD 19.150 23.307 71.124 |
| 31.03 |
| 318 735 UC IBD 41.010 23.307 3.453 |
| 11.44 |
| 319 736 CD IBD 14.224 23.307 142.655 |
| 72.03 |
| 320 737 NON IBD NON IBD 5.788 23.307 -0.628 9.91 |
| 321 738 NON IBD NON IBD 10.776 23.307 1.255 |
| 6.48 |
| 322 739 UC IBD 14.840 23.307 6.403 |
| 5.87 |
| 323 740 NON IBD NON IBD 14.286 23.307 5.116 |
| 18.83 |
| 324 741 CD IBD 57.389 23.307 113.308 |
| 205.71 |
| 325 742 UC IBD 33.744 23.307 4.018 |
| 7.41 |
| 326 743 UC IBD 26.478 23.307 2.291 |
| 8.67 |
| 327 744 NON IBD NON IBD 11.576 23.307 0.220 |
| 10.87 |
| 328 745 UC IBD 36.392 23.307 -0.157 2.48 |
| 329 746 IBS NON IBD 19.150 23.307 3.013 |
| 4.6 |
| 330 747 NON IBD NON IBD 15.702 23.307 0.471 |
| 9.29 |
| 331 748 CD IBD 18.350 23.307 0.471 |
| 10.68 |
| 332 752 UC IBD 86.084 23.307 9.856 |
| 5.17 |
| 333 753 CD IBD 19.212 23.307 71.030 |
| 31.03 |
| 334 754 UC IBD 23.584 23.307 3.986 |
| 2.52 |
| 335 755 CD IBD 57.143 23.307 18.613 |
| 41.49 |
| 336 756 CD IBD 19.951 23.307 210.264 |
| 157.55 |
| 337 758 CD IBD 11.761 23.307 53.233 |
| 64.84 |
| 338 760 UC IBD 35.160 23.307 17.420 |
| 33.03 |
| 339 761 CONTROL NON IBD 26.152 20.930 1.682 |
| 7.67 |
| 340 762 UC IBD 47.260 20.930 2.548 |
| 7.98 |
| 341 763 CD IBD 29.016 20.930 40.987 |
| 10.6 |
| 342 764 CONTROL NON IBD 24.159 20.930 2.035 |
| 6.94 |
| 343 767 IBS NON IBD 17.995 20.930 38.776 |
| 27.37 |
| 344 768 NON IBD NON IBD 17.435 20.930 14.245 |
| 33.41 |
| 345 769 CD IBD 19.614 20.930 1.266 |
| 2.37 |
| 346 770 UC IBD 52.179 20.930 2.964 |
| 5.22 |
| 347 771 UC IBD 52.428 20.930 11.489 |
| 21.22 |
| 348 773 UC IBD 38.854 20.930 26.678 |
| 31.49 |
| 349 802 NON IBD NON IBD 18.182 20.930 7.435 |
| 15.91 |
| 350 803 CD IBD 42.403 20.930 82.583 |
| 89.88 |
| 351 804 NON IBD NON IBD 16.438 20.930 1.234 |
| 8.52 |
| 352 805 CD IBD 63.263 20.930 34.321 |
| 75.73 |
| 353 806 CD IBD 20.735 20.930 46.803 |
| 65.15 |
| 354 809 UC IBD 81.133 20.930 1.009 |
| 4.48 |
| 355 810 CD IBD 25.716 20.930 104.054 |
| 62.92 |
| 356 811 CD IBD 15.567 20.930 76.334 |
| 64.38 |
| 357 813 UC IBD 51.681 20.930 0.641 |
| 8.23 |
| 358 814 CD IBD 18.182 20.930 26.598 |
| 68.68 |
| 359 816 CD IBD 24.844 20.930 10.880 |
| 27.28 |
| 360 817 UC IBD 33.686 20.930 1.394 |
| 9.78 |
| 361 820 UC IBD 31.756 20.930 5.704 |
| 13.15 |
| 362 823 UC IBD 23.848 20.930 1.058 |
| 4.5 |
| 363 828 CD IBD 19.676 20.930 35.347 |
| 105.74 |
| 364 830 UC IBD 24.222 20.930 1.859 |
| 9.63 |
| 365 831 UC IBD 72.727 20.930 4.278 |
| 13.47 |
| 366 832 UC IBD 85.367 20.930 10.399 |
| 8.05 |
| 367 833 UC IBD 18.804 20.930 2.115 |
| 19.43 |
| 368 834 UC IBD 26.588 20.930 4.198 |
| 14.73 |
| 369 835 CD IBD 13.138 20.930 5.405 |
| 55.37 |
| 370 836 CD IBD 16.376 20.930 39.533 |
| 23.98 |
| 371 837 NON IBD NON IBD 6.351 20.930 12.597 |
| 36.86 |
| 372 839 CD IBD 16.252 20.930 118.140 |
| 145.7 |
| 373 841 UC IBD 15.567 20.930 1.619 |
| 7.7 |
| 374 862 CD IBD 20.152 15.673 93.814 |
| 111.16 |
| 375 863 CD IBD 38.431 15.673 2.410 |
| 34.92 |
| 376 874 CD IBD 9.496 15.673 29.593 |
| 62.83 |
| 377 875 CD IBD 50.111 15.673 12.453 |
| 12.57 |
| 378 905 CD IBD 34.909 15.673 5.316 |
| 5.59 |
| 379 906 CD IBD 34.641 15.673 66.149 |
| 56.76 |
| 380 914 CD IBD 33.972 15.673 12.199 |
| 14.98 |
| 381 929 CD IBD 9.184 15.673 32.325 |
| 19.78 |
| 382 930 UC IBD 33.839 15.673 5.651 |
| 16.69 |
| 383 939 UC IBD 114.044 15.673 2.825 |
| 7.33 |
| 384 940 IBS NON IBD 2.987 15.673 3.656 |
| 4.45 |
| 385 942 CD IBD 13.286 15.673 81.427 |
| 51.87 |
| 386 943 CD IBD 4.547 15.673 3.990 |
| 5.91 |
| 387 945 CD IBD 12.158 11.390 6.856 |
| 21.79 |
| 388 947 CD IBD 5.410 11.390 26.312 |
| 69.17 |
| 389 948 UC IBD 20.790 11.390 6.387 |
| 8.38 |
| 390 949 UC IBD 132.219 11.390 3.508 |
| 9.13 |
| 391 950 CD IBD 9.666 11.390 3.575 |
| 7.12 |
| 392 951 UC IBD 45.350 11.390 13.484 |
| 33.78 |
| 393 954 CD IBD 11.307 11.390 39.743 |
| 38.29 |
| 394 955 UC IBD 23.647 11.390 1.366 |
| 8.29 |
| 395 957 UC IBD 18.541 11.390 4.874 |
| 9.17 |
| 396 959 IBS NON IBD 10.274 11.390 4.379 |
| 4.36 |
| 397 961 UC IBD 11.611 11.390 0.951 |
| 17.45 |
| 398 962 UC IBD 12.584 11.390 0.589 |
| 7.12 |
| 399 963 UC IBD 19.939 11.390 2.906 |
| 30.43 |
| 400 964 UC IBD 38.116 11.390 3.696 |
| 11.70 |
| 401 965 IBS NON IBD 6.505 11.390 1.861 |
| 2.82 |
| 402 966 IBS NON IBD 6.505 11.390 11.061 |
| 10.45 |
| 403 967 CD IBD 7.234 11.390 27.009 |
| 102.39 |
| 404 968 NON IBD NON IBD 18.541 11.390 4.111 |
| 9.36 |
| 405 969 IBS NON IBD 12.462 11.390 6.709 |
| 21.60 |
| 406 970 CD IBD 29.119 11.390 76.995 |
| 40.16 |
| 407 971 CD IBD 19.453 11.390 91.885 |
| 74.55 |
| 408 972 CD IBD 11.672 11.390 43.198 |
| 104.83 |
| 409 973 UC IBD 29.240 11.390 2.384 |
| 9.65 |
| 410 974 UC IBD 21.033 11.390 6.909 |
| 9.93 |
| 411 979 CD IBD 26.261 11.390 88.431 |
| 166.66 |
| 412 1003 NON IBD NON IBD 4.216 10.493 3.878 |
| 13.01 |
| 413 1004 CD IBD 3.972 10.493 7.574 |
| 18.13 |
| 414 1005 CD IBD 27.767 10.493 12.185 |
| 44.66 |
| 415 1007 CD IBD 11.755 10.493 0.763 |
| 9.02 |
| 416 1008 UC IBD 38.346 10.493 25.683 |
| 13.29 |
| 417 1009 UC IBD 5.310 10.493 4.031 |
| 11.30 |
| 418 1011 NON IBD NON IBD 5.918 10.493 3.791 |
| 4.64 |
| 419 1014 CD IBD 33.847 10.493 3.901 |
| 7.71 |
| 420 1017 NON IBD NON IBD 2.351 10.493 0.069 |
| 2.87 |
| 421 1020 UC IBD 10.215 10.493 3.846 |
| 17.73 |
| 422 1022 CONTROL NON IBD 6.972 10.493 3.878 |
| 16.03 |
| 423 1025 UC IBD 40.940 10.493 2.541 |
| 34.58 |
| 424 1026 CONTROL NON IBD 11.715 10.493 7.574 |
| 59.29 |
| 425 1029 NON IBD NON IBD 4.580 10.493 6.991 |
| 11.24 |
| 426 1031 CONTROL NON IBD 5.432 10.493 14.558 |
| 7.66 |
| 427 1032 NON IBD NON IBD 1.500 10.493 3.063 |
| 12.84 |
| 428 1034 WEGENER'S NON IBD 76.976 10.493 9.463 |
| 25.99 |
| 429 1035 CONTROL NON IBD 9.972 10.493 15.726 |
| 32.48 |
| 430 1040 UC IBD 28.334 10.493 3.118 |
| 15.00 |
| 431 1042 UC IBD 70.409 10.493 4.477 |
| 11.70 |
| 432 1043 UC IBD 16.214 10.493 7.595 |
| 10.05 |
| 433 1044 UC IBD 36.927 10.493 1.538 |
| 8.51 |
| 434 1045 UC IBD 20.916 10.493 6.139 |
| 14.55 |
| 435 1046 UC IBD 31.658 10.493 3.390 |
| 8.51 |
| 436 1047 UC IBD 121.078 10.493 41.656 |
| 22.63 |
| 437 1048 UC IBD 46.500 10.493 7.142 |
| 12.84 |
| 438 1048 UC IBD 46.453 10.493 3.970 |
| 12.84 |
| 439 1049 UC IBD 110.661 10.493 38.580 |
| 25.70 |
| 440 1050 UC IBD 21.970 10.493 3.063 |
| 3.50 |
| 441 1051 UC IBD 44.751 10.493 1.058 |
| 18.46 |
| 442 1052 CD IBD 29.179 11.390 7.183 |
| 39.51 |
| 443 1054 UC IBD 9.688 10.493 4.162 |
| 19.40 |
| 444 1055 UC IBD 7.621 10.493 43.662 |
| 35.22 |
| 445 1056 UC IBD 61.411 10.493 21.796 |
| 79.56 |
| 446 1057 CONTROL NON IBD 8.188 10.493 1.799 |
| 12.03 |
| 447 1058 UC IBD 16.052 10.493 12.842 |
| 66.21 |
| 448 1059 UC IBD 10.230 12.074 16.852 |
| 19.23 |
| 449 1061 UC IBD 29.967 12.074 5.439 |
| 48.63 |
| 450 1062 UC IBD 6.820 12.074 0.247 |
| 5.33 |
| 451 1063 UC IBD 3.148 12.074 3.214 |
| 8.46 |
| 452 1064 UC IBD 10.303 12.074 2.362 |
| 10.49 |
| 453 1066 UC IBD 40.852 12.074 8.680 |
| 14.01 |
| 454 1067 UC IBD 11.148 12.074 1.112 |
| 3.96 |
| 455 1068 UC IBD 11.803 12.074 4.299 |
| 6.26 |
| 456 1069 UC IBD 15.279 12.074 1.387 |
| 2.47 |
| 457 1070 UC IBD 5.508 12.074 1.195 |
| 7.53 |
| 458 1071 UC IBD 2.885 12.074 6.167 |
| 10.71 |
| 459 1072 UC IBD 70.230 12.074 28.554 |
| 18.46 |
| 460 1073 UC IBD 25.508 12.074 3.379 |
| 9.56 |
| 461 1074 UC IBD 48.131 12.074 8.721 |
| 12.97 |
| 462 1075 CONTROL NON IBD 6.557 12.074 5.700 |
| 13.02 |
| 463 1076 CONTROL NON IBD 3.410 12.074 0.4.81 |
| 6.87 |
| 464 1077 UC IBD 29.605 11.390 7.760 |
| 8.08 |
| 465 1080 CONTROL NON IBD 3.607 12.074 2.884 |
| 15.44 |
| 466 1081 UC IBD 45.115 12.074 2.474 |
| 9.45 |
| 467 1106 UC IBD 13.435 11.390 7.664 |
| 6.92 |
| 468 1107 UC IBD 45.410 11.390 10.507 |
| 20.88 |
| 469 1109 UC IBD 19.149 11.390 10.739 |
| 19.07 |
| 470 1110 UC IBD 137.812 11.390 18.189 |
| 12.55 |
| 471 1111 UC IBD 20.182 11.390 8.038 |
| 49.95 |
| 472 1112 UC IBD 63.100 11.390 1.109 |
| 19.12 |
| 473 1114 CONTROL NON IBD 12.280 11.390 25.052 |
| 10.00 |
| 474 1135 CONTROL NON IBD 7.086 11.931 2.270 |
| 11.25 |
| 475 1136 CONTROL NON IBD 11.118 11.931 1.062 |
| 67.24 |
| 476 1139 CONTROL NON IBD 9.346 11.931 21.020 |
| 34.18 |
| 477 1141 CONTROL NON IBD 11.973 11.931 5.227 |
| 5.49 |
| 478 1143 CONTROL NON IBD 19.181 11.931 24.465 |
| 8.46 |
| 479 1201 CONTROL NON IBD 8.430 11.931 11.091 |
| 40.82 |
| 480 1207 CONTROL NON IBD 13.561 11.931 5.115 |
| 22.20 |
| 481 1208 CONTROL NON IBD 13.500 11.931 15.423 |
| 26.45 |
| 482 1209 CONTROL NON IBD 19.976 11.931 5.271 |
| 28.89 |
| 483 1227 UC IBD 112.645 11.931 1.200 |
| 14.79 |
| 484 1229 CONTROL NON IBD 12.523 11.931 36.391 |
| 23.02 |
| 485 1230 CONTROL NON IBD 5.987 11.931 3.079 |
| 9.38 |
| 486 1303 CONTROL NON IBD 7.880 11.931 4.410 |
| 12.96 |
| 487 1305 CONTROL NON IBD 8.369 11.931 1.527 |
| 2.13 |
| 488 1307 CONTROL NON IBD 9.835 11.931 9.982 |
| 17.15 |
| 489 1308 UC IBD 20.648 11.931 19.794 |
| 10.29 |
| 490 1309 CONTROL NON IBD 11.546 11.931 20.420 |
| 50.30 |
| 491 1323 CONTROL NON IBD 11.607 11.931 4.345 |
| 9.15 |
| 492 1326 CONTROL NON IBD 17.288 11.931 5.454 |
| 9.53 |
| 493 1330 CONTROL NON IBD 5.070 11.931 4.736 |
| 20.73 |
| 494 1334 CONTROL NON IBD 11.057 11.931 1.253 |
| 2.90 |
| 495 1408 CONTROL NON IBD 12.034 11.931 30.089 |
| 13.11 |
| 496 1413 CONTROL NON IBD 7.941 11.931 3.719 |
| 5.11 |
| 497 1419 CONTROL NON IBD 11.057 11.931 3.732 |
| 82.70 |
| 498 1420 CONTROL NON IBD 7.636 11.931 5.493 |
| 9.53 |
| 499 1427 UC IBD 97.373 11.931 6.080 |
| 17.61 |
| 500 1436 CONTROL NON IBD 21.503 11.931 1.840 |
| 12.50 |
| 501 1440 UC IBD 35.858 11.931 36.443 |
| 15.85 |
| 502 1501 UC IBD 109.312 12.074 5.806 |
| 11.36 |
| 503 1505 UC IBD 131.869 12.074 2.518 |
| 5.95 |
| 504 1506 CD IBD 10.623 12.074 85.530 |
| 46.27 |
| 505 1507 CD IBD 2.230 12.074 101.096 |
| 46.27 |
| 506 1511 CD IBD 3.738 12.074 5.741 |
| 10.29 |
| 507 1514 UC IBD 159.279 12.074 3.510 |
| 28.28 |
| 508 1517 CD IBD 43.869 12.074 16.310 |
| 13.95 |
| 509 1519 UC IBD 79.475 12.074 4.684 |
| 11.28 |
| 510 1603 CD IBD 2.623 12.074 98.904 |
| 39.94 |
| 511 1604 CD IBD 8.197 12.074 47.651 |
| 74.09 |
| 512 1605 CD IBD 10.557 12.074 16.127 |
| 30.03 |
| 513 1607 CD IBD 10.623 12.074 10.626 |
| 11.28 |
| 514 1608 CD IBD 3.213 12.074 6.971 |
| 27.82 |
| 515 1610 CD IBD 6.557 12.074 0.368 |
| 0.91 |
| 516 1611 CD IBD 6.623 12.074 97.540 |
| 77.97 |
| 517 1613 CD IBD 15.157 22.230 73.203 |
| 33.61 |
| 518 1614 CD IBD 22.410 22.230 16.171 |
| 16.23 |
| 519 1616 CD IBD 8.365 22.230 96.712 |
| 63.34 |
| 520 1618 CD IBD 12.164 22.230 1.454 |
| 3.28 |
| 521 1619 CD IBD 7.406 22.230 54.002 |
| 27.13 |
| 522 1620 CD IBD 17.114 22.230 6.346 |
| 36.52 |
| 523 1623 CONTROL NON IBD 13.738 22.230 11.442 |
| 23.18 |
| 524 1625 CD IBD 21.757 22.230 0.000 |
| 8.78 |
| 525 1627 CD IBD 61.704 22.230 0.775 |
| 5.27 |
| 526 1628 CD IBD 6.293 22.230 8.330 |
| 13.11 |
| 527 1632 CONTROL NON IBD 8.135 22.230 97.214 |
| 108.62 |
| 528 1634 CD IBD 6.370 22.230 67.536 |
| 16.35 |
| 529 1636 CONTROL NON IBD 7.483 22.230 0.489 |
| 6.87 |
| 530 1638 CONTROL NON IBD 7.713 22.230 2.786 |
| 8.78 |
| 531 1639 CD IBD 12.394 22.230 0.802 |
| 9.56 |
| 532 1640 CD IBD 8.596 22.230 33.564 |
| 34.06 |
| 533 1712 UC IBD 154.144 22.230 0.000 |
| 3.39 |
| 534 1713 CD IBD 5.871 22.230 3.030 |
| 11 |
| 535 1714 CD IBD 11.704 22.230 44.775 |
| 27.16 |
| 536 1728 CONTROL NON IBD 13.085 22.230 1.807 |
| 11.04 |
| 537 1805 UC IBD 92.748 22.230 -1.957 7.73 |
| 538 1811 UC IBD 25.058 22.230 5.762 |
| 21.73 |
| 539 1812 CD IBD 12.164 22.230 3.085 |
| 19.08 |
| 540 1813 CONTROL NON IBD 5.833 22.230 -0.544 19.2 |
| 541 1817 CONTROL NON IBD 4.375 22.230 0.272 |
| 5.77 |
| 542 1914 CONTROL NON IBD 15.925 22.230 10.096 |
| 8.86 |
| 543 1939 UC IBD 11.282 22.230 1.508 |
| 7.96 |
| 544 1945 CD IBD 11.972 22.230 19.541 |
| 24.85 |
| 545 2002 UC IBD 52.724 22.230 57.236 |
| 18.73 |
| 546 2010 UC IBD 35.125 11.931 8.588 |
| 10.65 |
| 547 2017 CD IBD 11.484 11.931 22.761 |
| 16.54 |
| 548 2025 UC IBD 31.643 11.931 -0.707 7.06 |
| 549 2027 UC IBD 92.059 11.931 7.949 |
| 23.60 |
| 550 2034 CD IBD 11.240 11.931 76.996 |
| 32.31 |
| 551 2037 CD IBD 102.671 20.016 2.603 |
| 11.74 |
| 552 2048 CD IBD 19.565 20.016 34.830 |
| 117.25 |
| 553 2049 UC IBD 125.839 20.016 3.284 |
| 18.30 |
| 554 2050 UC IBD 63.043 20.016 3.231 |
| 4.37 |
| 555 2152 CD IBD 32.547 20.016 10.201 |
| 556 2203 CD IBD 13.292 20.016 5.345 |
| 10.97 |
| 557 2205 CD IBD 14.286 20.016 26.638 |
| 31.35 |
| 558 2228 CONTROL NON IBD 10.870 20.016 3.668 |
| 9.05 |
| 559 2232 CD IBD 28.882 20.016 118.812 |
| 110.53 |
| 560 2233 UC IBD 49.814 20.016 8.245 |
| 15.17 |
| 561 2234 UC IBD 76.211 20.016 0.000 |
| 5.56 |
| 562 2236 CONTROL NON IBD 8.944 20.016 1.310 |
| 5.48 |
| 563 2237 CD IBD 120.124 20.016 20.437 |
| 47.36 |
| 564 2240 CD IBD 30.932 20.016 135.179 |
| 97.72 |
| 565 2241 CD IBD 24.845 20.016 108.489 |
| 53.72 |
| 566 2243 CD IBD 10.870 20.016 66.498 |
| 20.22 |
| 567 2244 CD IBD 22.609 20.016 2.725 |
| 3.60 |
| 568 2250 UC IBD 78.634 20.016 8.611 |
| 14.85 |
| 569 2252 UC IBD 74.161 20.016 6.393 |
| 15.77 |
| 570 2253 UC IBD 26.025 20.016 0.838 |
| 6.24 |
| 571 2254 CD IBD 14.907 20.016 4.175 |
| 7.37 |
| 572 2256 CD IBD 18.012 20.016 3.843 |
| 17.01 |
| 573 2257 CD IBD 31.553 20.016 54.603 |
| 33.55 |
| 574 2259 CD IBD 20.000 20.016 132.804 |
| 120.82 |
| 575 2261 CD IBD 28.199 20.016 114.690 |
| 91.47 |
| 576 2270 NON IBD NON IBD 20.559 20.016 11.651 |
| 7.57 |
| 577 2303 CD IBD 7.329 20.016 4.000 |
| 13.81 |
| 578 2314 UC IBD 93.665 20.016 6.847 |
| 5.48 |
| 579 2315 CONTROL NON IBD 11.242 20.016 8.157 |
| 6.93 |
| 580 2318 UC IBD 5.031 20.016 50.515 |
| 22.18 |
| 581 2358 CD IBD 14.806 25.355 17.782 |
| 23.70 |
| 582 2364 UC IBD 32.524 25.355 4.873 |
| 8.85 |
| 583 2368 UC IBD 10.194 25.355 60.541 |
| 67.69 |
| 584 2406 UC IBD 94.053 25.355 11.913 |
| 15.33 |
| 585 2407 CD IBD 15.291 25.355 13.205 |
| 57.89 |
| 586 2408 CD IBD 40.473 25.355 20.035 |
| 18.69 |
| 587 2420 CD IBD 179.612 25.355 4.419 |
| 22.38 |
| 588 2422 CD IBD 11.529 25.355 4.603 |
| 16.09 |
| 589 2427 UC IBD 13.046 25.355 3.217 |
| 10.93 |
| 590 2429 UC IBD 285.012 25.355 4.795 |
| 20.69 |
| 591 2435 UC IBD 56.675 25.355 5.945 |
| 12.14 |
| 592 2437 CD IBD 12.197 25.355 2.896 |
| 22.49 |
| 593 2438 UC IBD 165.473 25.355 2.283 |
| 26.24 |
| 594 2439 CD IBD 142.901 25.355 17.880 |
| 37.63 |
| 595 2442 UC IBD 19.478 25.355 112.226 |
| 159.45 |
| 596 2447 UC IBD 80.886 25.355 0.291 |
| 23.01 |
| 597 2451 CD IBD 26.699 25.355 14.003 |
| 75.11 |
| 598 2452 UC IBD 22.209 25.355 16.210 |
| 17.02 |
| 599 2453 UC IBD 123.544 25.355 51.601 |
| 51.27 |
| 600 2454 CD IBD 22.573 25.355 3.034 |
| 24.06 |
| 601 2456 UC IBD 20.813 25.355 110.694 |
| 64.62 |
| 602 2464 CD IBD 62.864 25.355 64.961 |
| 146.93 |
| 603 2466 CD IBD 21.905 25.355 31.592 |
| 88.16 |
| 604 2467 UC IBD 41.080 25.355 3.080 |
| 23.54 |
| 605 2473 CD IBD 21.516 12.657 21.327 |
| 15.52 |
| 606 2475 UC IBD 21.890 12.657 17.497 |
| 54.27 |
| 607 2477 UC IBD 46.716 12.657 3.049 |
| 23.09 |
| 608 2503 UC IBD 30.112 12.657 1.593 |
| 10.72 |
| 609 2506 UC IBD 71.276 12.657 0.919 |
| 16.19 |
| 610 2507 UC IBD 76.241 12.657 7.783 |
| 35.01 |
| 611 2509 CD IBD 13.454 12.657 84.036 |
| 80.88 |
| 612 2510 CD IBD 16.231 12.657 107.722 |
| 138.91 |
| 613 2511 CD IBD 16.551 12.657 25.770 |
| 23.46 |
| 614 2514 UC IBD 24.933 12.657 0.000 |
| 12.59 |
| 615 2516 UC IBD 104.965 12.657 0.797 |
| 10.49 |
| 616 2517 UC IBD 38.174 12.657 2.237 |
| 32.01 |
| 617 2520 CD IBD 23.599 12.657 9.622 |
| 21.06 |
| 618 2521 UC IBD 40.790 12.657 88.096 |
| 88.98 |
| 619 2522 UC IBD 24.372 13.499 1.731 |
| 21.96 |
| 620 2533 UC IBD 39.349 12.657 1.593 |
| 15.22 |
| 621 2535 CD IBD 9.343 12.657 0.950 |
| 6.45 |
| 622 2537 UC IBD 69.301 12.657 0.000 |
| 4.57 |
| 623 2538 CD IBD 4.849 20.382 2.978 |
| 7.5 |
| 624 2540 CD IBD 10.358 12.657 102.712 |
| 132.08 |
| 625 2541 UC IBD 8.596 12.657 2.405 |
| 16.49 |
| 626 2542 UC IBD 38.334 12.657 5.592 |
| 19.85 |
| 627 2543 UC IBD 150.721 20.382 7.531 |
| 24.76 |
| 628 2555 CD IBD 96.003 20.382 0.915 |
| 14.87 |
| 629 2557 UC IBD 16.710 20.382 1.774 |
| 11.76 |
| 630 2559 UC IBD 140.105 20.382 5.912 |
| 20.75 |
| 631 2560 CD IBD 9.174 20.382 7.573 |
| 15.7 |
| 632 2561 CD IBD 5.177 20.382 3.533 |
| 23.51 |
| 633 2562 CD IBD 6.750 20.382 10.206 |
| 8.71 |
| 634 2563 CD IBD 17.369 20.382 16.258 |
| 6.78 |
| 635 2564 UC IBD 8.257 20.382 0.479 |
| 5.05 |
| 636 2569 CD IBD 29.030 20.382 5.870 |
| 24.2 |
| 637 2570 UC IBD 9.183 12.657 2.365 |
| 11.89 |
| 638 2572 UC IBD 109.877 12.657 83.784 |
| 39.83 |
| 639 2573 UC IBD 71.223 12.657 3.773 |
| 39.56 |
| 640 2575 CD IBD 15.750 12.657 6.461 |
| 12.45 |
| 641 2576 UC IBD 65.350 12.657 1.478 |
| 16.39 |
| 642 2577 CD IBD 11.426 12.657 101.788 |
| 22.75 |
| 643 2578 CD IBD 21.244 13.499 82.630 |
| 51.45 |
| 644 2579 UC IBD 68.830 13.499 0.338 |
| 4.36 |
| 645 2601 UC IBD 76.555 13.499 9.333 |
| 14.25 |
| 646 2602 CD IBD 5.253 13.499 45.861 |
| 31.54 |
| 647 2605 UC IBD 129.703 13.499 1.408 |
| 7.75 |
| 648 2607 UC IBD 66.242 13.499 3.125 |
| 7.05 |
| 649 2608 UC IBD 16.261 13.499 4.842 |
| 7.12 |
| 650 2609 CD IBD 13.055 13.499 26.675 |
| 34.92 |
| 651 2610 CD IBD 7.532 13.499 2.717 |
| 10.58 |
| 652 2611 UC IBD 13.210 13.499 1.182 |
| 8.78 |
| 653 2612 CD IBD 146.505 13.499 3.041 |
| 20.06 |
| 654 880585 CONTROL NON IBD 12.536 8.283 1.244 |
| 28.8184 |
| 655 890037 CONTROL NON IBD 5.364 8.283 1.168 |
| 13.5446 |
| 656 890361 CONTROL NON IBD 6.356 8.283 1.206 |
| 3.8904 |
| 657 890550 CONTROL NON IBD 3.907 8.283 8.517 |
| 8.9337 |
| 658 890581 CONTROL NON IBD 14.985 8.283 2.450 |
| 4.755 |
| 659 890622 CONTROL NON IBD 9.271 8.283 2.902 |
| 4.1786 |
| 660 890627 CONTROL NON IBD 17.318 8.283 31.242 |
| 22.9106 |
| 661 900097 CONTROL NON IBD 7.910 9.503 2.713 |
| 8.5014 |
| 662 900146 CONTROL NON IBD 5.726 9.503 0.735 |
| 7.7809 |
| 663 900224 CONTROL NON IBD 19.540 9.503 1.865 |
| 10.6628 |
| 664 900329 CONTROL NON IBD 9.504 9.503 1.489 |
| 2.7377 |
| 665 900421 CONTROL NON IBD 7.202 9.503 14.754 |
| 12.1037 |
| 666 900450 CONTROL NON IBD 6.907 9.503 0.471 |
| 4.3227 |
| 667 900452 CONTROL NON IBD 6.789 9.503 1.131 |
| 17.8674 |
| 668 900482 CONTROL NON IBD 11.452 9.503 6.425 |
| 19.1642 |
| 669 900504 CONTROL NON IBD 12.220 9.503 0.343 |
| 2.8184 |
| 670 900659 CONTROL NON IBD 14.699 9.503 4.250 |
| 11.9503 |
| 671 900709 CONTROL NON IBD 16.588 9.503 1.202 |
| 10.0338 |
| 672 900748 CONTROL NON IBD 7.792 9.503 1.524 |
| 4.735 |
| 673 910039 CONTROL NON IBD 4.723 9.503 3.713 |
| 5.2987 |
| 674 910042 CONTROL NON IBD 4.959 9.503 3.155 |
| 30.5524 |
| 675 910056 CONTROL NON IBD 13.872 9.503 1.760 |
| 13.3032 |
| 676 910095 CONTROL NON IBD 14.876 9.503 0.515 |
| 11.7249 |
| 677 910101 CONTROL NON IBD 13.991 9.503 2.576 |
| 30.5524 |
| 678 910104 CONTROL NON IBD 17.119 9.503 3.069 |
| 3.6076 |
| 679 910108 CONTROL NON IBD 9.622 9.503 1.717 |
| 6.3134 |
| 680 910156 CONTROL NON IBD 3.011 9.503 1.996 |
| 6.5388 |
| 681 910214 CONTROL NON IBD 6.966 9.503 0.622 |
| 7.779 |
| 682 910217 CONTROL NON IBD 4.604 9.503 0.880 |
| 5.9751 |
| 683 910220 CONTROL NON IBD 12.633 9.503 3.069 |
| 15.558 |
| 684 910234 CONTROL NON IBD 12.279 9.503 5.216 |
| 117.1364 |
| 685 910561 CONTROL NON IBD 11.393 9.503 5.646 |
| 2.2099 |
| 686 920028 CONTROL NON IBD 15.821 9.503 1.695 |
| 11.5172 |
| 687 920056 CONTROL NON IBD 12.102 9.503 2.284 |
| 4.2073 |
| 688 920142 CONTROL NON IBD 9.858 9.503 5.789 |
| 36.2091 |
| 689 920184 CONTROL NON IBD 9.792 11.138 7.163 |
| 6.3545 |
| 690 920258 CONTROL NON IBD 4.036 11.138 4.077 |
| 9.0969 |
| 691 920260 CONTROL NON IBD 6.706 11.138 4.481 |
| 6.2207 |
| 692 920264 CONTROL NON IBD 9.080 11.138 3.343 |
| 3.5451 |
| 693 920302 CONTROL NON IBD 6.944 11.138 4.555 |
| 6.1538 |
| 694 920346 CONTROL NON IBD 12.226 11.138 10.670 |
| 52.3745 |
| 695 920448 CONTROL NON IBD 10.564 11.138 4.353 |
| 5.3511 |
| 696 930031 CONTROL NON IBD 12.997 11.138 1.635 |
| 5.9531 |
| 697 930182 CONTROL NON IBD 4.392 11.138 5.197 |
| 12.9097 |
| 698 930184 CONTROL NON IBD 2.967 11.138 2.204 |
| 31.505 |
| 699 930219 CONTROL NON IBD 11.157 11.138 1.157 |
| 12.1739 |
| 700 930222 CONTROL NON IBD 11.039 11.138 14.123 |
| 18.2608 |
| 701 930225 CONTROL NON IBD 4.392 11.138 2.755 |
| 10.903 |
| 702 930228 CONTROL NON IBD 2.849 11.138 4.463 |
| 7.4916 |
| 703 930243 CONTROL NON IBD 10.148 11.138 0.680 |
| 9.3645 |
| 704 930266 CONTROL NON IBD 4.273 11.138 9.605 |
| 16.321 |
| 705 930268 CONTROL NON IBD 11.988 11.138 14.509 |
| 14.9163 |
| 706 930516 CONTROL NON IBD 3.680 11.138 14.527 |
| 5.5518 |
| 707 940106 CONTROL NON IBD 7.596 11.138 1.451 |
| 3.4782 |
| 708 950795 CONTROL NON IBD 3.442 11.138 10.854 |
| 9.6321 |
| 709 950940 CONTROL NON IBD 1.068 11.138 1.157 |
| 4.0133 |
| 710 860211 CONTROL NON IBD 9.489 11.205 4.615 |
| 10.0094 |
| 711 860214 CONTROL NON IBD 7.688 11.205 0.671 |
| 3.0217 |
| 712 870018 CONTROL NON IBD 7.147 11.205 0.378 |
| 22.0018 |
| 713 880055 CONTROL NON IBD 6.907 11.205 0.378 |
| 3.4938 |
| 714 880071 CONTROL NON IBD 4.384 11.205 5.538 |
| 7.7431 |
| 715 880626 CONTROL NON IBD 11.832 11.205 3.923 |
| 25.118 |
| 716 890061 CONTROL NON IBD 4.745 11.205 0.441 |
| 1.8885 |
| 717 890163 CONTROL NON IBD 8.468 11.205 5.496 |
| 31.1614 |
| 718 890308 CONTROL NON IBD 8.724 8.754 0.399 |
| 2.2662 |
| 719 890353 CONTROL NON IBD 15.608 8.754 3.147 |
| 6.3267 |
| 720 890362 CONTROL NON IBD 7.003 8.754 1.575 |
| 14.1772 |
| 721 890516 CONTROL NON IBD 12.819 8.754 1.877 |
| 29.9578 |
| 722 890519 CONTROL NON IBD 9.318 8.754 1.381 |
| 3.4599 |
| 723 890523 CONTROL NON IBD 3.323 8.754 4.078 |
| 13.2489 |
| 724 890529 CONTROL NON IBD 21.899 8.754 1.251 |
| 2.8691 |
| 725 900560 CONTROL NON IBD 6.915 8.558 4.746 |
| 22.1097 |
| 726 900606 CONTROL NON IBD 5.230 8.558 0.604 |
| 26.4978 |
| 727 900608 CONTROL NON IBD 14.991 8.558 0.820 |
| 6.5822 |
| 728 910164 CONTROL NON IBD 19.349 8.558 36.980 |
| 13.924 |
| 729 920551 CONTROL NON IBD 8.731 10.984 9.250 |
| 8.0612 |
| 730 920552 CONTROL NON IBD 11.369 10.984 8.989 |
| 42.5 |
| 731 920584 CONTROL NON IBD 5.088 10.984 1.024 |
| 14.4786 |
| 732 920748 CONTROL NON IBD 9.381 11.947 29.980 |
| 49.2873 |
| 733 921032 CONTROL NON IBD 10.727 11.947 0.422 |
| 4.051 |
| 734 930026 CONTROL NON IBD 6.867 11.947 4.518 |
| 2.7006 |
| 735 930055 CONTROL NON IBD 6.499 11.947 6.928 |
| 23.0307 |
| 736 930077 CONTROL NON IBD 10.791 11.947 0.086 |
| 26.2565 |
| 737 930130 CONTROL NON IBD 7.848 11.947 0.964 |
| 4.201 |
| 738 930138 CONTROL NON IBD 14.654 11.947 1.406 |
| 7.5018 |
| 739 930230 CONTROL NON IBD 5.886 11.947 3.112 |
| 7.4268 |
| 740 930252 CONTROL NON IBD 16.048 13.486 1.667 |
| 12.378 |
| 741 930288 CONTROL NON IBD 11.916 13.486 0.321 |
| 5.0262 |
| 742 930300 CONTROL NON IBD 12.994 13.486 1.847 |
| 10.3525 |
| 743 930446 CONTROL NON IBD 13.713 13.486 3.412 |
| 13.6909 |
| 744 930559 CONTROL NON IBD 9.566 11.473 0.796 |
| 29.5436 |
| 745 930666 CONTROL NON IBD 5.489 11.473 8.599 |
| 17.324 |
| 746 930804 CONTROL NON IBD 8.987 10.373 0.994 |
| 2.2428 |
| 747 930838 CONTROL NON IBD 9.590 10.373 24.645 |
| 18.1497 |
| 748 930875 CONTROL NON IBD 6.553 12.139 1.754 |
| 6.9209 |
| 749 930877 CONTROL NON IBD 6.382 12.139 0.456 |
| 22.1751 |
| 750 930924 CONTROL NON IBD 8.091 12.139 4.438 |
| 7.6977 |
| 751 930925 CONTROL NON IBD 9.174 12.139 1.123 |
| 9.5338 |
| 752 930977 CONTROL NON IBD 7.806 12.139 1.772 |
| 11.0169 |
| 753 86-0034 S CONTROL NON IBD 8.367 10.951 6.390 |
| 16.0142 |
| 754 86-0074 S CONTROL NON IBD 14.490 10.951 3.465 |
| 18.6239 |
| 755 86-0085 S CONTROL NON IBD 5.306 9.812 2.776 |
| 10.1486 |
| 756 86-0126 S CONTROL NON IBD 14.552 9.115 1.036 |
| 11.0536 |
| 757 87-0005 S CONTROL NON IBD 11.293 10.951 14.048 |
| 12.9369 |
| 758 87-0022 S CONTROL NON IBD 2.734 10.815 2.955 |
| -2.397 |
| 759 87-0068 S CONTROL NON IBD 6.064 10.815 4.183 |
| 4.9438 |
| 760 87-0092 S CONTROL NON IBD 10.544 10.951 5.361 |
| 23.9855 |
| 761 87-0159 S CONTROL NON IBD 14.959 11.895 1.408 |
| 9.736 |
| 762 87-0292 S CONTROL NON IBD 8.186 9.812 2.916 |
| 7.8651 |
| 763 87-0294 S CONTROL NON IBD 6.620 9.812 1.611 |
| 10.1612 |
| 764 88-0250 S CONTROL NON IBD 4.274 11.895 1.001 |
| 4.1642 |
| 765 88-0280 S CONTROL NON IBD 9.456 10.951 5.224 |
| 75.0296 |
| 766 88-0397 S CONTROL NON IBD 6.361 12.672 2.793 |
| 5.5443 |
| 767 88-0448 S CONTROL NON IBD 8.097 10.815 3.349 |
| -4.1947 |
| 768 88-0555 S CONTROL NON IBD 10.971 10.815 15.416 |
| 1.4981 |
| 769 88-0658 S CONTROL NON IBD 10.340 10.951 4.371 |
| 9.4899 |
| 770 88-0662 S CONTROL NON IBD 18.035 9.115 1.376 |
| 4.9228 |
| 771 89-0683 S CONTROL NON IBD 12.932 11.895 4.715 |
| 10.1466 |
| 772 90-0136 S CONTROL NON IBD 12.585 10.951 10.076 |
| 23.0614 |
| 773 90-0180 S CONTROL NON IBD 9.674 10.815 1.784 |
| 146.1423 |
| 774 90-0218 S CONTROL NON IBD 10.166 12.672 2.099 |
| 11.2495 |
| 775 90-0233 S CONTROL NON IBD 15.872 15.179 7.081 |
| 18.9512 |
| 776 90-0255 S CONTROL NON IBD 17.951 15.179 3.666 |
| 13.5234 |
| 777 90-0261 S CONTRDL NON IBD 22.229 9.467 18.784 |
| 33.6706 |
| 778 90-0286 S CONTROL NON IBD 7.692 12.816 0.377 |
| 1.319 |
| 779 90-0302 S CONTROL NON IBD 8.590 9.812 5.422 |
| 4.137 |
| 780 90-0315 S CONTROL NON IBD 19.154 9.115 5.736 |
| 7.0458 |
| 781 90-0335 S CONTROL NON IBD 9.826 9.115 2.699 |
| 5.338 |
| 782 90-0703 S CONTROL NON IBD 7.669 12.672 30.082 |
| 25.7934 |
| 783 90-0734 S CONTROL NON IBD 19.593 10.815 2.018 |
| 6.0674 |
| 784 91-0267 S CONTROL NON IBD 28.027 10.951 3.604 |
| 18.8612 |
| 785 91-0484 S CONTROL NON IBD 6.660 10.815 9.683 |
| 11.2359 |
| 786 92-1001 S CONTROL NON IBD 6.013 9.812 5.477 |
| 2.3917 |
| 787 92-329 S CONTROL NON IBD 10.821 12.816 4.353 |
| 5.7156 |
| 788 92-404 S CONTROL NON IBD 90.045 9.812 3.555 |
| 3.6638 |
| 789 92-407 S CONTROL NON IBD 12.279 9.812 1.016 |
| 3.5661 |
| 790 92-466 S CONTROL NON IBD 5.104 9.812 6.863 |
| 8.549 |
| 791 92-702 S CONTROL NON IBD 5.328 10.815 5.953 |
| 8.6891 |
| 792 92-809 S CONTROL NON IBD 12.164 11.895 0.831 |
| 12.6099 |
| 793 92-832 S CONTROL NON 1BD 11.973 10.951 6.217 |
| 8.5978 |
| 794 92-900 S CONTROL NON IBD 10.450 10.803 2.375 |
| 20.0000 |
| 795 92-9721 S CONTROL NON IBD 5.272 10.278 3.140 |
| 11.7276 |
| 796 92-988 S CONTROL NON IBD 14.490 9.115 15.896 |
| 33.2256 |
| 797 93-0487 S CONTROL NON IBD 16.849 17.859 9.829 |
| 4.6658 |
| 798 93-0509 S CONTROL NON IBD 3.077 12.816 0.363 |
| 2.0517 |
| 799 93-0654 S CONTROL NON IBD 27.709 12.980 13.160 |
| 13.4641 |
| 800 93-0741 S CONTROL NON IBD 10.338 17.859 1.483 |
| 12.6984 |
| 801 93-0746 S CONTROL NON IBD 8.388 9.812 4.745 |
| 8.6468 |
| 802 93-0768 S CONTROL NON IBD 10.410 12.816 1.165 |
| 3.4359 |
| 803 93-0996 S CONTROL NON IBD 7.291 10.815 2.077 |
| -2.0224 |
| 804 93-1001 S CONTROL NON IBD 19.728 10.951 2.873 |
| 22.8351 |
| 805 93-1010 S CONTROL NON IBD 4.629 9.467 4.406 |
| 8.3891 |
| 806 93-1039 S CONTROL NON IBD 5.029 9.467 1.478 |
| 2.4096 |
| 807 94-0017 S CONTROL NON IBD 26.288 10.815 3.042 |
| 17.2284 |
| 808 94-0083 S CONTROL NON IBD 6.015 10.803 7.010 |
| 7.5294 |
| 809 94-0095 S CONTROL NON IBD 9.990 15.179 1.394 |
| 4.5078 |
| 810 94-0104 S CONTROL NON IBD 4.343 9.467 39.441 |
| 25.3458 |
| 811 94-0143 S CONTROL NON IBD 23.365 12.672 15.723 |
| 20.8211 |
| 812 94-0181 S CONTROL NON IBD 11.567 9.115 1.585 |
| 8.185 |
| 813 94-0189 S CONTROL NON IBD 7.967 12.672 0.220 |
| 3.695 |
| 814 94-0228 S CONTROL NON IBD 13.767 10.278 2.342 |
| 10.0392 |
| 815 94-0237 S CONTROL NON IBD 13.205 11.895 7.395 |
| 20.9384 |
| 816 94-0245 S CONTROL NON IBD 9.744 12.816 2.684 |
| 11.2848 |
| 817 94-0301 S CONTROL NON IBD 15.676 12.980 3.221 |
| 14.8173 |
| 818 94-0308 S CONTROL NON IBD 9.200 9.467 2.001 |
| 11.3788 |
| 819 94-0384 S CONTROL NON IBD 14.513 17.859 1.507 |
| 12.0428 |
| 820 94-0459 S CONTROL NON IBD 30.599 10.815 8.688 |
| 12.1348 |
| 821 94-0466 S CONTROL NON.IBD 12.752 12.980 1.527 |
| 1.6914 |
| 822 94-0467 S CONTROL NON IBD 4.686 9.467 19.113 |
| 27.9668 |
| 823 94-0550 S CONTROL NON IBD 5.314 9.467 2.554 |
| 35.4306 |
| 824 94-0569 S CONTROL NON IBD 7.551 12.672 5.684 |
| 13.2985 |
| 825 94-0635 S CONTROL NON IBD 9.333 12.816 7.079 |
| 25.5243 |
| 826 94-0655 S CONTROL NON IBD 8.939 10.238 1.987 |
| 4.4011 |
| 827 94-0719 S CONTROL NON IBD 16.347 12.980 17.939 |
| 0.6089 |
| 828 94-0727 S CONTROL NON IBD 6.171 9.467 5.929 |
| 9.5046 |
| 829 94-0794 S CONTROL NON IBD 16.836 15.179 3.807 |
| 18.8592 |
| 830 95-0029 S CONTROL NON IBD 4.101 10.278 9.122 |
| 38.5245 |
| 831 95-0033 S CONTROL NON IBD 9.959 10.278 2.324 |
| 16.5195 |
| 832 95-0073 S CONTROL NON IBD 9.888 15.179 4.261 |
| 3.4958 |
| 833 95-0108 S CONTROL NON IBD 14.400 9.467 2.330 |
| 3.1236 |
| 834 95-0109 S CONTROL NON IBD 20.086 12.980 16.321 |
| 20.0947 |
| 835 95-0114 S CONTROL NON IBD 14.503 15.179 4.935 |
| 9.4756 |
| 836 95-0181 S CONTROL NON IBD 4.114 9.467 2.479 |
| 7.0058 |
| 837 95-0191 S CONTROL NON IBD 12.697 10.803 3.206 |
| 6.9019 |
| 838 95-0192 S CONTROL NON IBD 13.943 9.467 6.173 |
| 14.4434 |
| 839 95-0225 S CONTROL NON IBD 16.826 12.980 4.229 |
| 2.7063 |
| 840 95-0275 S CONTROL NON IBD 4.469 10.238 4.968 |
| 7.1428 |
| 841 95-0338 S CONTROL NON IBD 5.858 10.278 10.159 |
| 8.9533 |
| 842 95-0554 S CONTROL NON IBD 2.855 10.803 1.977 |
| 17.3333 |
| 843 95-0558 S CONTROL NON IBD 11.891 10.238 1.758 |
| 17.8932 |
| 844 95-0684 S CONTROL NON IBD 29.657 9.467 0.862 |
| 26.1269 |
| 845 95-0716 S CONTROL NON IBD 8.006 12.980 8.977 |
| 9.4046 |
| 646 95-0880 S CONTROL NON IBD 17.029 9.467 14.742 |
| 12.0515 |
| 847 95-0887 S CONTROL NON IBD 7.095 12.980 10.992 |
| 30.9201 |
| 848 95-1012 S CONTROL NON IBD 13.998 12.980 9.420 |
| 30.5818 |
| 849 95-1038 S CONTROL NON IBD 15.159 17.859 7.097 |
| 26.2295 |
| 850 95-1077 S CONTROL NON IBD 14.066 17.859 2.858 |
| 4.7919 |
| 851 96-0107 S CONTROL NON IBD 9.197 10.278 3.339 |
| 7.9215 |
The individuals described in Table 1 were classed in one of several disease or control categories. As shown in Table 2, of the 851 total patients, 433 (50.88%) were in the IBD category and 418 (49.12%) were in the Non-IBD category. All serum samples were tested by neutrophil ELISA and for immunoglobulin G and immunoglobulin A antibodies to mannan from Saccharomyces cerevisiae uvarum as described in Example I. Neutrophil ELISA positive samples were additionally analyzed by immunofluorescence assay with neutrophil substrate, followed by DNase treatment for immunofluorescence positive samples that show a perinuclear pattern. No other measurements were made on the samples.
| TABLE 2 |
| Inflammatory Bowel Disease Database |
| Disease or Control |
| Category Number of Patients Percent of Total |
| IBD Category |
| Crohn's disease* 218 25.62 |
| Ulcerative 212 24.91 |
| colitis* |
| Ulcerative 3 0.35% |
| colitis/PSC* |
| IBD Subtotal 433 50.88 |
| Non-IBD category |
| Disease control* 60 7.05 |
| Non-IBD* 35 4.11 |
| IBS* 22 2.59 |
| Wegener's 1 0.12 |
| granulomatosis* |
| Control 300 35.35 |
| Non-IBD subtotal 418 49.12 |
| Total 851 100 |
| *verified by colonoscopy, radiology and/or histology |
B. Simultaneous Variation of ANCA, ASCA-IgA and ASCA-IgG Cut-Off Values
The three different ELISA cut-off values for ANCA reactivity, ASCA-IgA reactivity and ASCA-IgG reactivity were varied simultaneously. Base cut-off values were determined as follows:
To determine the base cut-off value for ANCA reactivity, a panel of twenty verified negative control samples was used with a calibrator with a defined ELISA Unit (EU) value. The base positive/negative cut-off for each ELISA run was defined as the optical density (OD) of the Calibrator minus the mean (OD) value for the panel of twenty negatives (plus 2 standard deviations) times the EU value of the Calibrator. The base cut-off value for ANCA reactivity was therefore about 10 to 20 EU, with any patient sample having an average EU value greater than the base cut-off marked as ELISA positive for ANCA reactivity. Similarly, a patient sample having an average EU value is less than or equal to the base cut-off was determined to be negative for ANCA reactivity.
To determine the base cut-off value for ASCA-IgA and ASCA-IgG, single point calibrators having fixed EU values were used. OD values for patient samples were compared to the OD value for the Calibrators and multiplied by the Calibrator assigned values. The base cut-off value for the ASCA-IgA ELISA was 20 EU. The base cut-off value for the ASCA-IgG ELISA was 40 EU.
Using this existing set of test data for 851 patients having IBD status determined by colonoscopy or radiology or both or who were asymptomatic controls, the three cut-off values were simultaneously adjusted to observe the effects on clinical parameters: sensitivity, specificity, negative predictive value, positive predictive value, and overall agreement. In particular, design of Experiments (DOE) methodology was used to simultaneously vary the three cut-off ELISA values and to determine the effects on the resulting clinical parameters of sensitivity, specificity, negative predictive value, positive predictive value and overall agreement. The DOE methodology is advantageous in that variables are tested in a nested array requiring fewer runs and identifying cooperative interactions among the three cut-off variables. Optimization software DOE Keep It Simple Statistically (KISS) was obtained from Air Academy Associates (Colorado Springs, Colo.) and used to assign experimental runs and perform the simultaneous equation calculations.
Cut-off values were varied as set forth in Table 3 below:
| TABLE 3 |
| Ranges of ANCA, ASCA-IgA and ASCA-IgG cut-off values |
| Low Cut-off Standard High Cut-off |
| ELISA Value Cut-off Value Value |
| ANCA 0.5X Standard 1.0X Standard 1.5X Standard |
| ASCA-IgA 10 EU 20 EU 30 EU |
| ASCA-IgG 20 EU 40 EU 60 EU |
A three variable (ANCA cut-off, ASCA-IgA cut-off, ASCA-IgG cut-off) and three level (low, middle, and high; see Table 3) central composite (CCD) factorial design experiment was conducted as follows. In each experiment listed, the cut-off values for each of the three ELISA tests were set as shown in the first column of Table 4. Analysis using the KISS program was made with all first, second and third order variables operable. The first experiment shown in row 1 of Table 4, for example, indicates a cut-off value of ANCA=0.5, ASCA-IgA=10, and ASCA-IgG=20. By comparison with these assigned cut-off values, the test results determined for all of the 851 samples in the data base were assigned as true positive, true negative, false positive, or false negative. Using these results and the clinically defined diagnosis, sensitivity, specificity, overall agreement, positive predictive value, and negative predictive value were calculated. Using the DOE KISS program, optimized sets of cut-off values for selected clinical parameters were calculated.
The clinical parameter results for each set of three cut-off variables are shown in Table 4. Although these results are the calculated points determined by the experimental design, clinical parameter results for any other set of cut-offs within the cut-off boundaries also can be calculated. The three dimensional test box determined by the extremes of the three variables defines the region in which testing was conducted. These results show that there is a continuum of solutions of clinical responses within the boundaries of the cut-off values and that the DOE methodology can be used to determine the sets of cut-off values which present the most useful clinical parameters for a particular patient population.
| TABLE 4 |
| Clinical Parameter Results from Simultaneous Variation |
| of ANCA, ASCA-IgA, and ASCA-IgG cut-off values X, Y, |
| and Z in a population with 50% disease prevalence |
| % Overall |
| Cut-offs* % Sens. % Spec. Agreement % PPV % NPV |
| 0.5/10/20 96.3 13.6 55.7 60.5 78.1 |
| 0.5/10/60 95.2 14.8 55.7 53.6 74.7 |
| 0.5/30/20 96.1 14.4 55.9 53.8 77.9 |
| 0.5/30/60 94.9 16.3 56.3 54.0 75.6 |
| 1.5/10/20 81.3 64.6 73.1 70.4 76.9 |
| 1.5/10/60 77.1 75.4 76.3 76.4 76.1 |
| 1.5/36/20 78.5 70.8 74.4 73.6 76.1 |
| 1.5/30/60 69.5 86.1 77.7 83.7 73.2 |
| 1.0/20/40 82.2 67.3 74.9 72.2 78.5 |
| 1.0/20/40 82.2 67.3 74.9 72.2 78.5 |
| 0.5/20/40 94.9 16.3 56.3 54.0 75.6 |
| 1.5/20/40 74.1 83.5 78.7 82.3 75.7 |
| 1.0/10/40 85.0 61.0 73.2 69.3 79.7 |
| 1.0/30/40 80.6 57.4 69.2 66.2 74.1 |
| 1.0/20/20 85.2 57.7 71.7 67.6 79.0 |
| 1.0/20/60 81.8 67.5 74.7 72.2 78.1 |
| *ANCA/ASCA-IgA/ASCA-IgG cut-offs |
The maximum possible sensitivity, specificity, negative predictive value, positive predictive value and overall agreement within the range of ANCA values (0.5× to 1.5× standard); ASCA-IgA values (10 To 30 ELISA units) and ASCA-IgG values (20 to 60 ELISA units) were determined with the entire 851 person database having an IBD disease prevalence of 50%. The results are shown in Table 5.
| TABLE 5 |
| Maximum possible clinical parameters in a population |
| having an IBD disease prevalence of 50% |
| Clinical Maximum |
| Parameters possible Cut-off values |
| (N = 851) correlation ANCA/ASCA-IgA/ASCA-IgG |
| Sensitivity 96.61% 0.50; 10.00; 20 |
| Specificity 87.57% 1.50; 24.48; 60 |
| Negative 80.25 0.90; 14.21; 20 |
| predictive value |
| Positive 84.54 1.50; 26.10; 60 |
| predictive value |
| Overall agreement 79.57% 1.46; 20.42; 60 |
The results shown in Table 5 give the maximum possible clinical parameters within the ranges of cut-off values explored in a population with an IBD disease prevalence of 50%. For example, the highest possible sensitivity is 96.61% and is obtained with an ANCA cut-off of 0.5, an ASCA-IgA cut-off of 10 EU, and an ASCA-IgG cut-off of 20 EU. At this high sensitivity, specificity is reduced, being only 13.16% at this cut-off. These results demonstrate that ANCA, ASCA-IgA and ASCA-IgG values can be determined to give maximum sensitivity, but that other cut-off values are needed to yield maximum specificity.
C. Determination of ANCA, ASCA-IgA and ASCA-IgG Cut-Off Values for High Sensitivity
Sensitivity is the fraction of all those with IBD who are diagnosed positive for IBD with the first step assay. Values were selected that produced a high sensitivity (90.3%) while still maintaining a relatively high specificity. In particular, 90.3% sensitivity was achieved by setting the ANCA cut-off at 0.7 multiplied by two standard deviations above the background value of ANCA-negative UC sera, ASCA-IgA cut-off at 12 EU and the ASCA-IgG cut-off at 60 EU (see Table 6). These cut-offs are distinct from the cut-offs used in the UC*Dx-1 and CD*Dx-1 assays, which are 1.0, 20, and 60, respectively.
| TABLE 6 |
| Evaulation of results with high sensitivity assay having |
| ANCA cut-off = 0.7, ASCA-IgA cut-off = 12 EU and |
| an ASCA-IgG cut-off = 60 EU. |
| True IBD True IBD |
| Positive Negative Totals |
| First step assay 391 262 653 |
| positive |
| First Step assay 42 156 198 |
| negative |
| Totals 433 418 851 |
With an ANCA cut-off of 0.7 multiplied by two standard deviations above the background value of ANCA-negative UC sera, an ASCA-IgA cut-off of 12 EU, and an ASCA-IgG cut-off of 60 EU, the specificity was determined to be 37.3%. Using these cut-off values and the entire 851 patient database (having an IBD disease prevalence of 50%), the negative predictive value was 78.8%, the positive predictive value was 59.9%, and the overall agreement was 64.3%. These data also can be modeled for an IBD prevalence of 15%, which represents the approximate IBD disease prevalence in a gastroenterologist's office population (see Table 7). In a population having an IBD prevalence of 15%, an ANCA cut-off of 0.7 multiplied by two standard deviations above the background value of ANCA-negative UC sera, an ASCA-IgA cut-off of 12 EU and an ASCA-IgG cut-off of 60 EU resulted in a negative predictive value of 95.6%, a positive predictive value of 20.3%, and overall agreement of 45.3%. The calculated performance at 15% IBD prevalence was confirmed by randomly choosing patients from the n=851 database to construct and analyze a new database (n=277) that had a 15% IBD prevalence.
| TABLE 7 |
| Clinical parameters with 50%, 15% and 1% IBD disease |
| prevalence for assays run with |
| an ANCA cut-off = 0.7, an ASCA-IgA cut-off = 12 EU |
| and an ASCA-IgG cut-off = 60 EU |
| Disease prevalence |
| Clinical parameter 50% 15% 1% |
| Sensitivity 90.3% 90.3% 90.3% |
| Specificity 37.3% 37.3% 37.3% |
| Negative 78.8% 95.6% 99.7% |
| predictive value |
| Positive 59.9% 20.3% 1.43% |
| predictive value |
| Overall agreement 64.3% 45.3% 37.8% |
This example demonstrates that the "First Step" diagnostic method can be used in combination with subsequent, specific diagnostic assays such as the UC*Dx-1 and CD*Dx-1 assays.
Samples which were positive by "First Step" analysis (including true positive and false positive samples) were subsequently tested by the UC*Dx-1 and CD*Dx-1 assays, which are specific for ulcerative colitis and Crohn's disease, respectively. The results are shown in Table 8.
| TABLE 8 |
| Results of reflex of samples positive by "First Step" |
| diagnosis to the UC*Dx-1 and CD*Dx-1 assays |
| True IBD True IBD |
| Positive Negative Totals |
| CD*Dx-1 and/or 287 38 325 |
| UC*Dx-1 Positive |
| CD*Dx-1 and/or 146 380 526 |
| UC*Dx-1 Negative |
| Totals 433 418 851 |
Together, reflex of samples positive by "First Step" analysis to the UC*Dx-1 and CD*Dx-1 performs with 66.3% sensitivity, 90.9% specificity, 72.2% negative predictive value, 88.3% positive predictive value and 78.4% overall agreement. These results indicate that subsequent analysis of positive samples can amplify on the initial "First Step" result. These results indicate that the "First Step" diagnostic can be used, if desired, in combination with a subsequent, more specific diagnostic method.
All journal article, reference and patent citations provided above, in parentheses or otherwise, whether previously stated or not, are incorporated herein by reference in their entirety.
Although the invention has been described with reference to the examples provided above, it should be understood that various modifications can be made without departing from the spirit of the invention. Accordingly, the invention is limited only by the claims.
Walsh, Michael J., Rose, Steven L.
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